Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.     

Factors affecting the success of oocyte transfer in a clinical program for subfertile mares

Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P > 0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than > or = 20 yr (mean ages +/- S.E.M. of 15.5 +/- 0.4 and 23.0 +/- 0.3 yr, respectively) tended (P = 0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.     

Luteal function in jennies following PGF(2alpha) treatment 3 days after ovulation

Native PGF(2alpha) and its analogs have been used in the horse mare to manipulate ovarian activity, primarily as luteolytic agents to induce estrus. Despite numerous studies on the effects of these luteolysins in the mare, to date only a single investigation has been conducted in the jenny. The aim of this study was to evaluate the response of the corpus luteum (CL) to a single dose of PGF(2alpha) given 3 days (72h) after ovulation and to establish the plasma progesterone (P4) profile from pre-treatment to post-treatment ovulation in the Martina Franca donkey. Twenty-two jennies were ultrasonographically monitored and treated 72h after the detection of ovulation with 0.075 mg i.m. of R-cloprostenol. From the day of ovulation until ovulation post-treatment, blood was collected daily for P4 determination by enhanced luminescence immunoassay. All the jennies except one, exhibited behavioral signs of PGF(2alpha)-induced estrus within 4 days of treatment lasting 5.4+/-1.16 days. Post-treatment ovulation was also hastened, reducing the interovulatory interval (9.6 days). In response to treatment, plasma P4 concentrations fell to estrus levels and then remained constant until the next ovulation in all but the non-responding animal. Our findings indicate that PGF(2alpha) treatment on Day 3 post-ovulation causes the functional regression of the CL in the jenny, reflected both by the rapid induction of estrus and ovulation and by an abrupt drop in circulating P4 concentrations.     

Phytoplankton assemblage at equilibrium in large and deep subalpine lakes: a case study from Lago Maggiore (N. Italy)

In 1996, we studied the phytoplankton seasonal succession in Lago Maggiore (N. Italy) through weekly sampling. Such a frequency enabled us to evaluate the changes of the phytoplankton assemblage in the light of the equilibrium and non-equilibrium theories. The distinct phases of changing of the species composition were identified separating the samples by means of cluster analysis and non-metric multidimensional scaling (NMDS). We recognised well distinct phytoplankton associations, whose seasonal succession followed a clear cyclic path throughout the year, with spring and summer phases respectively characterised by a rapid turn-over of the assemblages and by a relative stability. Moreover, we observed an increase of species number and Shannon-Wiener diversity during the spring, followed by a summer decline of the diversity in spite of an unchanged species number. Because of the dominance of the same few species for about two months during summer, coupled with small fluctuations of the total biomass, we could identify the summer assemblage as a steady state assemblage. The aim of the present contribution, although describing the whole seasonal succession, is to draw the attention towards the species composition at the steady state, taking into account the functional properties of the species involved. The possible role of the metalimnetic niche in selecting a particular summer assemblage in deep and large lakes will be discussed.     

An Apgar scoring system for routine assessment of newborn puppy viability and short-term survival prognosis

The Apgar scoring system is an easy and reliable method for evaluating both human and animal neonates. However, its use is not widespread in veterinary medicine. The current study assessed a modified Apgar scoring system for routine evaluation of newborn puppies. Heart rate, respiratory effort, reflex irritability, motility, and mucus color have been evaluated in the score. Specifically, we used 5 min after birth Apgar score to assess newborn viability and short-term survival prognosis, as well as related characteristics, in 193 puppies from 42 litters, 65 born by spontaneous delivery, 66 by assisted delivery, and 62 by cesarean section. The percentage of puppies that were dead 2 h after birth was higher in the 4 to 6 Apgar score group versus that in the 7 to 10 score group (P < 0.01) and in the 0 to 3 score group versus that in the 7 to 10 score group (P < 0.0001). Delivery method did not affect survival. There was a marked reduction in the number of puppies searching for the mammary gland in the 0 to 3 and 4 to 6 Apgar score groups compared with that in the 7 to 10 score group (P < 0.0001); there was a difference between the 0 to 3 and the 4 to 6 score groups as well (P < 0.05). Suckling/swallowing reflexes were present in fewer puppies in the 0 to 3 and 4 to 6 score groups compared with that in the 7 to 10 group (P < 0.0001), with no significant differences between the 0 to 3 and the 4 to 6 score groups.     

Levels of heat shock protein transcripts in normal follicles and ovarian follicular cysts

In the study, the gene expression of several heat shock proteins (HSPs) was determined in normal follicles and cystic follicles from cattle. A lower expression of HSP10 and HSP40 was observed in granulosa and theca cells of cysts compared to normal follicles. HSP27 was significantly less expressed in granulosa cells in cystic and large antral follicles than in other follicular categories. HSP60 and HSP90a expressions were highest in theca cells of cysts. However, HSP70 and HSP90b exhibited a lower expression in cysts than in healthy follicles.     

Peripartal plasma concentrations of 15-ketodihydro-PGF2α, cortisol, progesterone and 17-β-estradiol in Martina Franca jennies

The transition from intra- to extrauterine environment represents a very delicate phase, in which the successful coordination of maturation is strictly connected with several hormonal changes during the last weeks of gestation and at parturition. While the peripartal endocrinology in the mare has been deeply investigated, the peripartal hormonal changes in the jenny need further evaluation. The aim of this study is to evaluate the mean 15-ketodihydro-PGF_2α (PGFM), cortisol (C), progesterone (P4), and 17β-estradiol (E2) levels during the peripartal period in this species. Ten Martina Franca jennies, with normal gestational length and parturition, were enrolled. From each jenny, blood was collected twice a day from 10 d before to 7 d after parturition and from the plasma obtained PGFM, C, P4 and E2 were analyzed by RIA. Higher, constant PGFM concentrations were observed in the pre-foaling days compared to the decreasing levels detected the days after delivery, as previously observed in the mare. During the whole period of observation no significant differences in plasma C levels were detected. In contrast to the mare, P4 has always been detectable and the highest level found at −2.5 days was significantly different compared to samples obtained between −10 and −4.5 days and between 1.5 and 7 days after foaling. Finally, E2 showed higher concentrations before foaling, with the highest values between −3 and −1.5 days, decreasing only one day before foaling. A positive correlation was found between PGFM and P4, during the last 4 days of gestation, while a positive correlation between PGFM and E2 was observed during the prepartum. Despite some similarities with the mare exist, differences have been found in P4 and E2 profiles, underlining once more the differences in the physiology of this two species.     

Efficacy of hCG and GnRH for inducing ovulation in the jenny

Knowledge about management of ovulation in the donkey is limited compared to that in the horse. This experiment was designed to evaluate the efficacy of injecting single doses of lecirelin (a GnRH-analogue) or of hCG to induce ovulation in the jenny and to determine whether effects are dependent upon follicular diameter at time of injection. Ovarian activity and follicular growth were monitored by rectal ultrasonography. Jennies were randomly allotted to the following groups: Group GnRH, treated with 100 microg lecirelin; Group hCG, treated with 2500 IU hCG; Group C, untreated and monitored for spontaneous ovulation. Animals were also categorized into subgroups depending upon follicular diameter: 30-35 mm (GnRH-1, hCG-1 and C-1) or 36-40 mm (GnRH-2, hCG-2 and C-2). Jennies in the two hormone treatment groups did not differ significantly for time from treatment to ovulation, but there was a significant reduction in time to ovulation as follicle size at treatment increased. Jennies treated with either lecirelin or hCG had significantly smaller follicle size at ovulation than jennies in the Control groups that underwent spontaneous ovulation. Treatment groups did not differ significantly in the proportion of jennies that ovulated within 48 h of injection or between 25 and 48 h following injection. These results highlight the usefulness of lecirelin for induction and synchronization of ovulation in the jenny, particularly since it would avoid the risk of reduced hCG response in reproductive management programs in which that hormone was repeatedly used.     

Thioredoxin from the Indianmeal Moth Plodia interpunctella: Cloning and Test of the Allergenic Potential in Mice

Background/Objective

The Indianmeal moth Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1) has been identified.

Objective

The aim of this study was to identify further allergens and characterise these in comparison to Plo i 1.

Method

A cDNA library from whole adult P. interpunctella was screened with the serum of a patient with indoor allergy and IgE to moths, and thioredoxin was identified as an IgE-binding protein. Recombinant thioredoxin was generated in E. coli, and tested together with Plo i 1 and whole moth extracts in IgE immunoblots against a large panel of indoor allergic patients'' sera. BALB/c mice were immunised with recombinant thioredoxin and Plo i 1, and antibody production, mediator release from RBL cells, T-cell proliferation and cytokine production were measured.

Result

For the first time a thioredoxin from an animal species was identified as allergen. About 8% of the sera from patients with IgE against moth extracts reacted with recombinant P. interpunctella thioredoxin, compared to 25% reacting with recombinant Plo i 1. In immunised BALB/c mice, the recombinant allergens both induced classical Th2-biased immune responses such as induction IgE and IgG1 antibodies, upregulation of IL-5 and IL-4 and basophil degranulation.

Conclusion

Thioredoxin from moths like Plo i 1 acts like a classical Type I allergen as do the thioredoxins from wheat or corn. This clearly supports the pan-allergen nature of thioredoxin. The designation Plo i 2 is suggested for the new P. interpunctella allergen.