Three Finnish incontinentia pigmenti (IP) families with recombinations with the IP loci at Xq28 and Xp11

The locus (IP2) for the hereditary form of incontinentia pigmenti (IP) has been mapped to Xq28 by linkage analysis. We studied three IP families with polymorphic markers in the Xq28 region. In two families we observed recombination between the marker loci and IP. In the third family no crossing overs were seen and linkage to the Xq28 region could not be excluded. The other IP locus (IP1) has been mapped to Xp 11.21, because of sporadic cases of IP with X-chromosomal alterations involving Xp11.21. To check whether this locus is linked to IP in these families, we used polymorphic markers in the Xp11 region. In all three families recombinations were observed, thus excluding linkage to this locus in these IP families.     

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

Translocation (X;9)(p11;q34) in a girl with incontinentia pigmenti (IP): implications for the regional assignment of the IP locus to Xp11?

A t(X;9)(p11;q34) is reported in a girl with incontinentia pigmenti (IP). The X breakpoint is at p11.21. Although no similar case has been reported, this breakpoint may be significant insofar IP is considered an X-linked dominant mutation and could be of help in a specific X DNA probes study.     

Incontinentia pigmenti: Xp breakpoint is not the same in a case of r(X) and in X/autosome translocations

X-specific DNA probes were used to characterize the r(X) of a 45,X/46,X,r(X) female patient with Incontinentia pigmenti. It was found to be of maternal origin. Breakpoints were shown to be in or distal to p11.22 and between q12.2 and q13.1. When considering all known cases of Incontinentia pigmenti and X rearrangements at least four different break sites on the X have been shown.     

Replication timing properties across the pseudoautosomal region boundary and cytogenetic band boundaries on human distal Xp

The establishment of human chromosomal regions as distinct and characteristic domains has been demonstrated by the reproducible banding patterns observed on metaphase chromosomes as a result of various staining techniques. Although the exact molecular properties responsible for the patterns are not well understood, a general correlation has been established between the time of replication of a particular region of DNA and its banding characteristics. Using a replication timing assay based on fluorescence in situ hybridization patterns, we investigated replication timing properties across chromosomal regions with potentially distinct chromatin properties. Relative replication timing values were determined using cosmid DNA probes around the pseudoautosomal region boundary in Xp22.3 and the cytogenetic band boundary regions surrounding Xp22.2. Although we observed replication timing domains that were generally consistent with cytogenetic banding patterns, we did not find sharp replication timing boundaries at either the pseudoautosomal region boundary or at the cytogenetic band boundaries. Received: 6 September 1997; in revised form: 16 December 1997 / Accepted: 5 January 1998     

Trypanosoma cruzi: long-term sub-cultures in two different culture media do not confirm the existence of highly versatile multilocus genotypes

Trypanosoma cruzi Y reference strain is found in many laboratories under at least two highly distinct genotypes, A and B corresponding to the 'discrete typing units' T. cruzi IIb and T. cruzi IId, respectively. Previous work has reported reversible switches between these genotypes according to the culture media used in the experiments: genotype A would be associated with blood-enriched culture media, while genotype B would be associated with blood-free culture media. We tried to reproduce this observation, but used a different cloning method of individual organisms. Our cloning was verified visually under the microscope, while the previous studies relied on a cloning by dilution only. The subclones so obtained were submitted to long-term exposure to both media, and no change was observed in isoenzyme and random amplified polymorphic DNA genotypes. The discrepancy is probably explained by the cloning method: clones obtained from the previous method (dilution and plating) could come from several parasite cells while only one cell generates a clone when micro-manipulation is used.     

Studies to confirm the source of 11ß-hydroxyandrostenedione

In a longitudinal study of 82 children we found a gradual rise in median plasma concentrations of 11ß-hydroxyandrostenedione (11ß-OH-A4) from 2.5 to 6.4 nmol/1 during childhood which was similar in both sexes. This could reflect changes in adrenal function during the adrenarche and sexual maturation. Plasma concentrations of 11ß-OH-A4 in adults follow the patterns of cortisol secretion. In patients with diseases of the adrenal cortex, the plasma concentrations of 11ß-OH-A4 were consistent with the pathology of each condition. In women with polycystic ovaries (PCO) undergoing gonadotrophic stimulation for in vitro fertilization and embryo transfer, 11ß-OH-A4 (median = 3.8 nmol/l), testosterone and androstenedione, were raised when compared to women with normal ovaries (11ß-OH-A4 median = 2.6 nmol/l). Follicular fluid has concentrations of 11ß-OH-A4 six to twelve times greater than plasma levels and in women with PCO, 11ß-OH-A4 concentrations were lower than in women with normal ovaries, which is consistent with an inhibition of ovarian 11ß-hydroxylase. Granulosa cells in vitro demonstrated the production of 11ß-OH-A4 by side chain cleavage of cortisol. These data support an adrenal source for 11ß-OH-A4 but the raised plasma concentrations in women with polycystic ovary syndrome (PCOS) may reflect the excess androgen output from the ovary. 11ß-OH-A4 may therefore be an additional marker for ovarian dysfunction.     

Significance of cytogenetic studies in the preleukemias

In cytogenetic examinations made on 60 patients in preleukemic states an instable karyotype could be identified in 28% of all cases and anomalous clones in 12.3%. The dynamics of transition to acute leukemia was pursued in seven cases. The instability of the karyotype is conceived of as a sign of increased risk to developing leukemia and the presence of anomalous clones as an evidence of a leukemic process.     

Comparisons of treatment means when factors do not interact in two-factorial studies

Scientists in the fields of nutrition and other biological sciences often design factorial studies to test the hypotheses of interest and importance. In the case of two-factorial studies, it is widely recognized that the analysis of factor effects is generally based on treatment means when the interaction of the factors is statistically significant, and involves multiple comparisons of treatment means. However, when the two factors do not interact, a common understanding among biologists is that comparisons among treatment means cannot or should not be made. Here, we bring this misconception into the attention of researchers. Additionally, we indicate what kind of comparisons among the treatment means can be performed when there is a nonsignificant interaction among two factors. Such information should be useful in analyzing the experimental data and drawing meaningful conclusions.     

A somatic cell hybrid panel to facilitate identification of DNA sequences in the vicinity of the incontinentia pigmenti locus (IP1)

Somatic cell hybrids that retain derivative X chromosomes from women with sporadic incontinentia pigmenti (IP1) and de novo X/autosomal translocations with consistent breakpoints at Xp11.21 were constructed. An assembled hybrid panel was used to physically map DNA sequences in relationship to the IP breakpoint. DSX14 was found to map to region Xp11.21----p11.1. Regional assignments of 19 X-chromosomal loci were reviewed.     

In vitro cytogenetic studies of cypermethrin on human lymphocytes

Assessment of cytotoxicity and response to external factors like pesticides were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) or MTT assay, which measures mitochondrial metabolism in the entire cell culture and provides information about the percentage of cell survival. Utilizing the MTT assay, the cytotoxicity of cypermethrin was determined on lymphocyte cultures from human peripheral blood samples, the short-term lymphocyte cultures were incubated with various aliquots of the cypermethrin and the LC50 was found to be 33.6 microM. Lymphocytes treated with low-doses (1/10 of LC50) of cypermethrin showed an increase in the frequency of chromosomal aberrations and found to be significant. Karyotype analysis revealed more satellite associations and chromosomal breaks in cypermethrin treated samples. Low-doses of the pesticide also induced single-strand breaks in the DNA as assessed by comet assay. The pesticide caused increase in the comet tail length with increase in pesticide concentration, implicating genotoxicity in somatic cells. It is concluded that In vitro assays could give important information of the mechanism of toxicity at low dosages and impact on genetic material of human origin.     

A radiation hybrid map of the proximal short arm of the human X chromosome spanning incontinentia pigmenti 1 (IP1) translocation breakpoints.

Radiation hybrid mapping was used in combination with physical mapping techniques to order and estimate distances between 14 loci in the proximal region of the short arm of the human X chromosome. A panel of radiation hybrids containing human X-chromosomal fragments was generated from a Chinese hamster-human cell hybrid containing an X chromosome as its only human DNA. Sixty-seven radiation hybrids were screened by Southern hybridization with sets of probes that mapped to the region Xp11.4-Xcen to generate a radiation hybrid map of the area. A physical map of 14 loci was constructed based on the segregation of the loci in the hybrid clones. Using pulsed-field gel electrophoresis (PFGE) analyses and a somatic cell hybrid mapping panel containing naturally occurring X; autosome translocations, the order of the 14 loci was verified and the loci nearest to the X-chromosomal translocation breakpoints associated with the disease incontinentia pigmenti 1 (IP1) were identified. The radiation hybrid panel will be useful as a mapping resource for determining the location, order, and distances between other genes and polymorphic loci in this region as well as for generating additional region-specific DNA markers.     

Direct binding studies do not support the existence of true alpha-adreno-receptors in rat white fat cells

The complexity of mitochondrial translation products in mouse liver and Ehrlich ascites tumor cells have been studied using a mitoplast system active in ³⁵S methionine incorporation. Electrophoretic analysis on gradient polyacrylamide-SDS gels and urea-SDS gels under highly dissociating conditions show that both of the mitochondrial systems synthesize about 22 polypeptides. Many of these ³⁵S labeled products compare with the polypeptides predicted by the DNA sequence analysis data reported by Anderson $\text{et al.}$ (1).     

EPR studies show that all lanthanides do not have the same order of binding to calmodulin

Calmodulin, spin labeled at Tyr-99, has been titrated with the lanthanides La3+, Nd3+, Eu3+, Tb3+, Er3+ and Lu3+ as well as Ca2+ and Cd2+. The titration was monitored by EPR and changes in mobility of the spin label, due to binding into the labeled site and protein conformational change, were observed. Comparison of these titration curves with theoretical binding curves for the various calmodulin-metal species, show that different lanthanides have different high affinity sites. Three basic categories were observed, with Lu3+ and Er3+ behaving like Ca2+, Eu3+ and Tb3+ binding in the opposite order from Ca2+, and La3+ and Nd3+ different from either Ca2+ or Tb3+.