Marker chromosome identification by micro-FISH

Micro-FISH was used to elucidate the chromosomal origin of marker chromosomes in three patients. Ten copies of marker chromosomes were collected with microneedles from GTG banded metaphases, transferred to a collecting drop and amplified by means of DOP-PCR. The PCR products were labeled with biotin-14-dATP and used as FISH probes for hybridization to normal metaphase chromosomes and to metaphase chromosomes of the patients (reverse painting). With the generation of chromosome region-specific painting probes by PCR amplification of microdissected DNA and subsequent FISH it was possible to identify the marker chromosomes in all patients. One marker appeared to be derived from the centromere region of the X-chromosome and the proximal third of the long arm, one from the centromere region of chromosome 17 and one marker chromosome was identified as an isochromosome 18p.     

The gene for schnyder's crystalline corneal dystrophy maps to human chromosome 1p34.1-p36

Schnyder's crystalline corneal dystrophy (SCCD) is an autosomal dominant eye disease characterized by a bilateral clouding of the central cornea, arcus lipoides and/or visible crystalline deposits of cholesterol in the stroma. There is accumulation of phospholipid, unesterified cholesterol and cholesterol ester in the corneal stroma; this is believed to be due to an imbalance in the local factors affecting lipid/cholesterol transport or metabolism. The cellular mechanism of abnormal lipid transport and metabolism in SCCD is of interest due to its potential involvement in atherosclerosis, and its implications for the pathogenesis of cerebrovascular, coronary and peripheral vascular disease as well as corneal opacification. To determine the chromosomal location of the SCCD locus, genome-wide linkage analysis has been performed in two large Swede-Finn kindreds recently identified in central Massachusetts. After analysing 300 microsatellite markers > 90% of the genome was excluded from linkage to the SCCD locus. We now report the chromosomal assignment of the gene for SCCD in both families to be 1p34.1-p36; the maximum multipoint lod- score was 8.48 in the interval between D1S214 and D1S503. From haplotype analysis, the SCCD locus lies in the 16 cM interval between markers D1S2663 and D1S228. Several candidate genes for SCCD have been localized to the 1p34.1-p36 interval.      

A pilot study of the human chorionic gonadotrophin test for ovarian hyperandrogenism

A controlled clinical study was designed to investigate the value of human chorionic gonadotrophin (HCG) challenge as a test for functional ovarian hyperandrogenism. Dexamethasone administration was followed by 5000 IU HCG and blood samples for steroid hormone assay were obtained 0, 8, 16, and 24 h thereafter. Study subjects were normal women (n = 13); women with functional ovarian hyperandrogenism, defined by androgen excess, amenorrhoea and an increased 17-hydroxyprogesterone response to nafarelin (n = 6); and normal men (n = 4). The responses of 17-hydroxyprogesterone, androstenedione and testosterone to HCG in women with functional ovarian hyperandrogenism were significantly greater than in normal women. However, the 17-hydroxyprogesterone response to HCG in functional ovarian hyperandrogenism was significantly lower after HCG than after nafarelin. The oestradiol response was also significantly lower after HCG than nafarelin, although oestradiol concentration more than doubled in normal women as well as in women with functional ovarian hyperandrogenism. The responses to HCG confirm that functional ovarian hyperandrogenism abnormalities are luteinizing hormone (LH)-dependent. Therefore, the 17- hydroxyprogesterone response to HCG could represent a useful test for the diagnosis of ovarian hyperandrogenism. The lower 17- hydroxyprogesterone response to HCG than to nafarelin in functional ovarian hyperandrogenism suggests that a follicle-stimulating hormone (FSH)-responsive factor modulates thecal 17-hydroxyprogesterone secretion. The oestradiol response to HCG is consistent with HCG directly stimulating the oestradiol secretion by thecal cells.      

There are multiple regulators of expression throughout the MHC

We have previously reported the use of deletion mutants to investigate the location and function of new MHC genes. Several new examples of these will be reported using a cell panel of deletion mutants derived from a heterozygous parental cell (A2-B44-DR12-DQ3//A1-B8-DR13-DQ1) and selected for the loss of HLA-A2 following spontaneous mutation or treatment with x-rays or mutagens. The extent of the haplotypic loss of the mutants has been characterised by serology, by Block typing and by using multiple heterozygous CA repeat markers on 6p. The panel of deletion mutants has been used to screen serological reagents. It has been found that some antibodies suggest reduced levels of expression of HLA-B44 and B8 (AHS#6 6034, 6039) in a mutant following deletion in a region close to D6S248 but not including HLA-A. This has been confirmed by flowcytometry using a B8 MoAb. Following deletion of HLA-A2, the expression of HLA-B was apparently restored. Further AHS#6 serum 6051 was found to have undefined non HLA extra reactivity in addition to HLA A10 and B16. It reacted strongly with the original cell and all mutant panel cells except for two cells in which the deletion included a segment between D6S265 and D6S273 (includes HLA-B). With further deletions centromeric of D6S273, cytotoxicity was restored. These data imply that there are several unidentified up and down regulatory elements within the MHC which react in cis and trans combinations. Serological techniques and deletion mutant panels are informative for the localisation of HLA regulatory elements, new genes, cofactors and/or conformational elements affected by the deletions.     

Evidence for kinetoplast and nuclear DNA homogeneity in Trypanosoma evansi isolates

The kinetoplast DNA minicircles from 13 stocks of trypanosomes designated as Trypanosoma evansi were digested with various restriction enzymes. We also examined the distribution of restriction site polymorphisms in the nuclear DNA of 9 of these stocks, using 7 different variable surface glycoprotein (VSG) and non-VSG probes. Restricted kinetoplast DNA (kDNA) fragments of some of these strains were cloned into M13 or PUC 18 vectors and sequenced. The restriction and sequence mapping showed that most of T. evansi isolates belonged to the A1 and A2 types of Borst and to two new closely related types A3 and A4. A notable exception was RoTat 4/1 derived from a Sudanese stock which was found to display a characteristic brucei-like minicircle heterogeneity. The T. evansi minicircles analysed are not only homogeneous in sequence but also the region similar to the conserved region in Trypanosoma brucei and Trypanosoma equiperdum is flanked on its 5' end by a palindromic repeat of part of the conserved region. The highly conserved sequence GGGCGGT which appears to correspond to the initiation of synthesis of one of the Okazaki fragments contains an additional G and is located as in T. brucei and T. equiperdum about 73 bp 5' from the ORI. The nuclear DNA analysis confirms the kDNA study in that all the T. evansi stocks are members of a very homogeneous group in terms of sequence divergence. Moreover, our analysis also confirms that T. evansi is more closely related to the West African T. b. brucei and T. b. gambiense than to other African trypanosomes.     

Cytogenetic abnormalities in uterine myomas are associated with myoma size

Uterine leiomyomata (myomas) are associated with a variety of characteristic cytogenetic abnormalities. The significance of these chromosomal aberrations in the pathobiology of myomas remains to be determined. The present study investigated the relationship between myoma cytogenetic abnormalities and size. A total of 114 myoma specimens were obtained from 92 patients undergoing myomectomy or hysterectomy. The maximum diameter of each myoma was measured and a portion of each myoma obtained for cytogenetic analysis. Karyotypes were analysed and categorized as normal, abnormal (non-mosaic) or mosaic. Cytogenetic analyses revealed 73 (64%) normal, 20 (18%) abnormal (non-mosaic), and 21 (18%) mosaic karyotypes. Mean myoma diameter was 6.5+/-0.44 cm with a range of 0.4-27 cm. Differences between the mean myoma diameter of specimens with normal versus abnormal karyotypes was determined by the Kruskal-Wallis test. The mean myoma diameter among specimens with abnormal (non-mosaic) karyotypes was significantly greater than myomas with normal karyotypes (10.2+/- 5.9 versus 5.9+/-4.2 cm; P < 0.001). The proportion of abnormal (non- mosaic) karyotypes in myomas >6.5 cm was compared to myomas <6.5 cm by chi2-analysis; myomas >6.5 cm demonstrated a significantly higher proportion of abnormal (non-mosaic) karyotypes when compared to myomas <6.5 cm (75 versus 34%; P < 0.02). In summary, a significant relationship exists between clonal cytogenetic abnormalities and myoma size, suggesting that chromosomal abnormalities associated with individual myomas enhance myoma growth.      

Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism/obesity in Pima indians

The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala-->Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3'- untranslated region of exon 8 of UCP2 and a C-->T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 +/- 7 years), unrelated, full-blooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium ( P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.      

Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus

Summary After amplification by PCR, the 5 region of the genome of hog cholera virus (HCV) strain Alfort 187 was cloned and sequenced. The nucleotide and deduced amino acid sequences were compared with the ones of other pestiviruses. By in vitro translation experiments we were able to demonstrate the protease activity of the p 20 protein of HCV.     

Mechanism of the bactericidal action of myeloperoxidase: increased permeability of the Escherichia coli cell envelope.

A sensitive and highly reproducible assay was utilized to study in vitro interactions of Listeria monocytogenes with resident and activated macrophages. The technique is not compromised by extracellular events and can readily differentiate between the efficiency of ingestion and the postphagocytic fate of bacteria. Heat-labile factors in human or homologous serum markedly enhanced the phagocytosis of Listeria without noticeably affecting the intracellular fate of the microorganisms. The behavior of Listeria within macrophages cultivated from C57BL/6 and BALB/c mouse strains corresponded to previous reports of in vivo growth patterns in inbred mice. Thioglycolate- or caseinate-elicited macrophages, although highly phagocytic, were unable to prevent the proliferation of Listeria. A bactericidal macrophage population was derived from from C57BL/6 mice which had been immunized intraperitoneally with a sublethal dose of L. monocytogenes and subsequently boosted with heat-killed homologous organisms. Elicitation of immune animals produced an increase in the percentage of peroxidase-positive macrophages, but this activity could not be correlated with restriction of intracellular bacterial growth.