[Fatal epidermal necrosis due to methotrexate use]. / Fatale epidermale necrose door methotrexaatgebruik.

A 53-year-old patient with known psoriasis presented with painful erosive plaques in existing psoriasis lesions, ulcerations in the mouth, and leukopenia. She used 10 mg of methotrexate daily without folic acid prophylaxis. During admission she developed pancytopenia, liver failure and acute kidney injury. She died after developing toxic shock syndrome. We aim to describe the use and toxic effects of methotrexate, how to recognise toxicity clinically and histologically, and treatment options.

Effect of asynchronous transfer on bovine embryonic development and relationship with early cycle uterine proteome profiles.

The uterus provides the nurturing environment that supports the growth of the early preimplantation bovine conceptus. To determine critical time points of uterine influence, in vitro-produced Day 7 blastocysts were transferred into synchronous (Day 7) uteri and asynchronous uteri (Days 5 or 9). Embryo growth was evaluated 7 and 15 days after transfer and compared with that of embryos generated by AI. Conceptuses recovered from asynchronous Day 9 transfers were fourfold larger than synchronous transfer or gestational Day 14 AI conceptuses; by 15 days after transfer, differences were less marked. Two-dimensional gel electrophoresis was used to compare the histotroph protein composition of uterine luminal flushings (ULF) on Days 5 and 9 after oestrous to determine any protein differences that would promote embryo growth. The ULF were collected by serially flushing the uteri of the same heifers and mature cows at different times of the cycle. Ten proteins that differed in abundance between Day 5 and 9 were identified by mass spectrometry. Three, namely phosphoserine aminotransferase 1, purine nucleoside phosphorylase and aldose reductase, were verified by western blot analysis as more abundant on Day 9 (P<0.002). Myostatin was present in only in Day 9 ULF, whereas tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and legumain were only detected in Day 14 ULF. Although mature cows had lower progesterone concentrations on Days 5 and 14 (P<0.05) and tended to have less TIMP2 than heifer groups, no other protein differences were detected. Thus, the embryo growth-enhancing environment on Day 9 was associated with temporal changes in the expression of several proteins of the histotroph.

Cloning and expression of two mRNAs encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus.

The crustacean hyperglycemic hormone (CHH) of the X-organ sinus gland complex is a multifunctional neurohormone primarily involved in the regulation of blood sugar levels. HPLC analysis of lobster sinus glands revealed two CHH-immunoreactive groups, each consisting of two isoforms with identical amino acid sequences and molecular weights. In order to obtain more information concerning the number and sequences of preproCHHs, and to study their expression, we isolated two full-length cDNAs encoding two different CHH preprohormones. Both preprohormone structures consist of a signal peptide, a CHH-precursor-related peptide and a highly-conserved CHH peptide. Expression studies revealed that the X-organ is not the only source of CHH mRNA because the ventral nerve system also expresses this mRNA. Based on these findings and earlier studies on the effect of eyestalk ablation, implantation of thoracic/abdominal ganglia as well as the multifunctionality of CHH, we postulate that CHH, present in the ventral nerve system is a good candidate for a supplementary role in the control of reproduction and molting.

Acute effects of nicotine on attention and response inhibition.

Smoking is highly prevalent among patients with Attention Deficit Hyperactivity Disorder (ADHD). Previous studies using the reversed continuous performance task (R-CPT) have suggested that nicotine reduces inattention. Since especially adults with ADHD have been claimed to suffer from a core deficit in inhibitory control, this study aimed at determining whether nicotine improves response inhibition in addition to attention. Sixteen healthy regular smokers participated in a pre/post treatment design in which transdermal patches containing 7 and 21 mg nicotine per day were administered in a counterbalanced, double-blind manner. In a second study, patches containing 0 mg (placebo) and 21 mg per day were administered to a different group of regular smokers. For replication purposes, the R-CPT and the profile of mood states (POMS) were administered. Furthermore, a different version of the continuous performance task (CPT-AX) and the stop-signal task, traditionally used to measure response inhibition, were presented. The high dose of nicotine was found to relieve self-reported Depression in Study 1 and Fatigue in Study 2. Performance data indicated acute effects of nicotine on attention-related, but not on inhibition-related measures. Especially the comparison with placebo revealed decreases in reaction time and variability of responding. The results imply that patients with ADHD smoke to reduce inattention.

Cloned cattle derived from a novel zona-free embryo reconstruction system.

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.

Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle.

Several studies have shown that both quiescent and proliferating somatic donor cells can be fully reprogrammed after nuclear transfer (NT) and result in viable offspring. So far, however, no comparative study has conclusively demonstrated the relative importance of donor cell cycle stage on nuclear cloning efficiency. Here, we compare two different types of bovine fetal fibroblasts (BFFs) that were synchronized in G(0), G(1), and different phases within G(1). We show that for non-transgenic (non-TG) fibroblasts, serum starvation into G(0) results in a significantly higher percentage of viable calves at term than synchronization in early G(1) or late G(1). For transgenic fibroblasts, however, cells selected in G(1) show significantly higher development to calves at term and higher post-natal survival to weaning than cells in G(0). This suggests that it may be necessary to coordinate donor cell type and cell cycle stage to maximize overall cloning efficiency.

Long-term alteration of follicular steroid concentrations in relation to subclinical endometritis in postpartum dairy cows.

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.

Transcriptional analysis of the short segment of the feline herpesvirus type 1 genome and insertional mutagenesis of a unique reading frame.

Transcription mapping was performed in the short region of the feline herpesvirus type 1 (FHV-1) genome as a first approach to the functional analysis of open reading frames encoding the homologs of the herpes simplex virus type 1 (HSV-1) gD, gl, gE, US9, and probably also US8.5. All reading frames appeared to be transcribed. Transcripts were grouped into two nested RNA sets; namely, the coterminal transcripts of gD and gl and the coterminal transcripts of gE, US8.5, and US9. This situation was similar to that found in the equivalent region of HSV-1. The FHV-1 ORFs US8.5 and US9 have not been described previously. Sequence analysis and comparison of the putative polypeptide encoded by US8.5 revealed that this ORF was unique to FHV-1. However, US8.5 of FHV-1 might be functionally related to its positional homologs in HSV-1 and equine herpesvirus type 1. In all three viruses, US8.5 does not seem to be critical for virus propagation in cell culture. This was shown for FHV-1 by isolating a mutant containing an insertion in US8.5 and comparing its growth properties in cell culture to those of the parent virus G2620. With regard to US9, conscientious amino acid sequence alignment of FHV-1 US9 and homologs in related herpesviruses suggests that this particular protein could contribute to the virus infectivity in vivo. This speculation was based on the highly conserved C-terminus of US9, starting with a characteristic YYSES motif and followed by a nuclear target sequence and a transmembrane region.

Effects of follicular size of cytoplast donor on the efficiency of cloning in cattle.

In cattle, oocytes obtained from follicles smaller than 3 mm in diameter can undergo maturation in vitro, progressing to MII and undergoing fertilization, but are developmentally incompetent. Cytoplasts were prepared from in vitro matured oocytes aspirated from small (1-3 mm) or large (6-12 mm) follicles and fused to serum starved mural granulosa cells. Following activation, reconstructed embryos were cultured for 7 days and classified G1 to G4, before being processed for nuclei counting or transferred to synchronized recipients. Oocytes from small follicles had lower rates of polar body extrusion (59.6 vs. 69%; 731/1230 vs. 608/857) and fusion (71.4 vs. 78.8%; 360/497 vs. 364/465; P < 0.06). There were no differences in total rate of blastocysts development (60 vs. 59.8%; small vs. large), or any grade classification. A significant interaction was detected between follicle size and embryo grade with G3 embryos from small follicles having a greater cell number. Developmental competence of G1 and G2 embryos did not differ at day 27 (48 vs. 46%; 16/33 vs. 17/37; small vs. large). Although there were no differences in fetal size between the two groups, differences in allantois length (53 vs. 86 mm; small vs. large; P < 0.002) and allantois width (9.5 vs. 13 mm; small vs. large; P < 0.06) were seen. No differences in survival to term (2/13 in each group) were observed. These results indicate that cytoplasts from follicles of 1-3 and 6-12 mm in diameter are equally developmentally competent when used in a nuclear transfer procedure.