Chromosome fragility in river buffalo cows exposed to dioxins.

Fifty river buffalo (Bubalus bubalis, 2n = 50) cows reared in two different provinces of Campania (southern Italy) underwent cytogenetic investigations to ascertain possible differences in their chromosome stability. One group (Caserta province) was under legal sequestration due to the presence in the milk mass of higher mean values of dioxins [21.79 pg/g of fat as sum of polychloro-dibenzo-dioxins (PCDDs), polychloro-dibenzo-furans (PCDFs) and dioxin-like polychlorobiphenyls (DL-PCBs)] than both those permitted (6.0 pg/g of fat as WHO-TEQ) and those (1.3 pg/g of fat as WHO-TEQ) observed in the control group raised in Salerno province. Two types of peripheral blood cell cultures were performed: without (normal cultures for the chromosome abnormality (CA) test: chromatid breaks, chromosome breaks, fragments) and with the addition of BrdU for the sister chromatid exchange (SCE) test). The CA test revealed a significantly (P < 0.01) higher chromosome fragility in the exposed cows compared to the control. Indeed, mean values of CA/cell were 1.26 ± 1.15 in exposed cows and 0.37 ± 0.71 in the control. Mean SCE was higher in exposed cows (8.50 ± 3.35) than that (8.29 ± 3.51) found in the control but the difference was not significant. Comparison within the same group of cows at first (FL) and multiple (ML) lactations revealed significantly (P < 0.01) higher mean values of CA/cell in exposed ML-cows vs FL-cows while no statistical differences were found between ML-cows and FL-cows in the control farm. By contrast, significantly (P < 0.01) higher mean values of SCE were found in both groups of FL-cows versus ML-cows. Comparisons with other previous studied species (sheep and cattle) were also performed.

A rare case of centric fission and fusion in a river buffalo (Bubalus bubalis, 2n = 50) cow with reduced fertility.

A 5-year-old river buffalo cow underwent cytogenetic investigation since it had only one male offspring, apparently with normal body constitution, which died one month after birth. The female carrier had normal body conformation and internal sex adducts, as revealed by rectal palpation performed by a specialist veterinary practitioner. The cow was found to carry a complex and rare chromosome abnormality. Indeed, a centric fission of one river buffalo (BBU) chromosome 1 with a subsequent (or simultaneous) centric fusion of BBU1p with BBU23 was revealed by both RBA-banding and specific molecular markers of BBU1p (DEFB1) and BBU23 (ACTA2). CBA-banding revealed a pale, very small C-band in the der1 (BBU1q) and a prominent C-band on the new biarmed chromosome originated by rob(1p;23). Both telomeric probes and AgNOR staining confirmed the Robertsonian translocation (rob), both FITC-signals and the NORs (BBU23) being telomerically located. Furthermore, telomeric signals on der1 (BBU1q) indicate that these 2 chromosomal events may be the result of a reciprocal translocation which occurred between BBU1 and BBU23.

Frequency and distribution of rob(1;29) in eight Portuguese cattle breeds.

Cytogenetic investigations performed in eight Portuguese cattle breeds revealed the presence of rob(1;29) in both heterozygous and homozygous conditions in all, and five breeds, respectively, with variable percentages of carriers as follows: 41.0% in Arouquesa, 69.9% in Barrosa, 39.4% in Maronesa, 2.8% in Mirandesa, 8.5% in Marinhoa, 1.8% in Mertolenga, 21.3% in Raca Brava and 21.5% in Alentejana. CBA- and RBA-banding were performed to ascertain the chromosomes involved in the chromosome abnormality. A total of 1,626 animals were investigated. Reproductive parameters (number of calves per 100 cows) were higher in Mirandesa (80%) when compared with both Maronesa (75%) and Barrosa (70%) breeds, underlining that rob(1;29) reduces fertility in the carriers.

Changes in lipid composition and lipogenic enzyme activities in liver of lambs fed omega-6 polyunsaturated fatty acids.

Twenty-four lambs (Ovis aries) were used in a 45-day finishing study to evaluate the effects of feeding diets high in linoleic acid (C(18:2), omega-6) on liver lipid composition and on lipogenic enzyme activities in subcellular fractions of liver. Lambs were fed either a 5% safflower oil (SO, high linoleic acid) supplemented diet or a control diet without added oil. SO feeding caused a reduction in the amount of serum and liver triacylglycerols and cholesterol, whereas the level of phospholipids in both tissues was hardly affected. In liver of SO-treated lambs an increase in the levels of C(18:2) and arachidonic acid (C(20:4), omega-6), together with a simultaneous decrease of saturated fatty acids, was observed. In comparison to rat liver, rather low activities of enzymes in the pathway for de novo fatty acid synthesis, i.e. acetyl-CoA carboxylase and fatty acid synthase, were found in lamb-liver cytosol. Both enzyme activities, as well as those of the NADPH-furnishing enzymes, were significantly reduced by SO feeding. In contrast, microsomal and especially mitochondrial fatty acid chain elongation activity, the latter being much higher than that of rat liver, were significantly increased in SO-treated lambs. In these animals, a stimulation of triangle up(9)-desaturase activity was observed in liver microsomes.

FISH-mapping of LEP and SLC26A2 genes in sheep, goat and cattle R-banded chromosomes: comparison between bovine, ovine and caprine chromosome 4 (BTA4/OAR4/CHI4) and human chromosome 7 (HSA7).

Sheep (OAR), goat (CHI) and cattle (BTA) R-banded chromosome preparations, obtained from synchronized cell cultures, were used to FISH-map leptin (LEP) and solute carrier family 26 member 2 (SLC26A2) genes on single chromosome bands. LEP maps on OAR4q32 and CHI4q32, being the first assignment of this gene to these two species. SLC26A2 maps on BTA7q24, OAR5q24 and CHI7q24. This gene, too, was assigned for the fist time to both sheep and goat chromosomes, while it was more precisely localized on a single chromosome band in cattle. Improved cytogenetic maps of BTA4/OAR4/CHI4 were constructed and compared with HSA7 revealing five main conserved segments and complex chromosome rearrangements, including a centromere repositioning, differentiating HSA7 and BTA4/OAR4/CHI4.

Increased frequencies of both chromosome abnormalities and SCEs in two sheep flocks exposed to high dioxin levels during pasturage.

During the past four years several livestock farms (sheep, cattle and river buffalo) in the provinces of Naples and Caserta (southern Italy) have been unable to sell their milk and other dairy products due to the levels of dioxins (17 different types) present in the milk mass exceeding the value permitted [3 pg/g of fat, as human WHO 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) equivalent]. While some farms, especially those showing relatively low levels of dioxins, have managed to reduce the dioxins in the milk below the permitted threshold by changing the diet, many, especially sheep farms, have failed to do so. Indeed, about 12,000 head of cattle, river buffaloes and sheep have so far been culled. In the present study, 34 and 42 sheep from two herds raised in the province of Naples (Acerra municipality) and where high levels of dioxins (50.65 and 39.51 pg/g of fat, respectively) were found in the milk mass, were cytogenetically investigated and compared with 20 sheep (control) raised 80 km away from the exposed area. Increases of both chromosome abnormalities (gap, chromosome and chromatid breaks) (17 and 8 times higher in the two exposed herds, respectively) and sister chromatid exchanges (SCEs) were found in both herds when compared with the control, and the differences were highly significant (P<0.001). No statistical differences were found when comparing the frequencies of aneuploid cells of exposed animals (16.4 and 17.8%) and control (17.9%). Furthermore, high levels of mortality and abnormal foetuses were recorded in one of the two farms when compared with the control.

Chromosome evolution and improved cytogenetic maps of the Y chromosome in cattle, zebu, river buffalo, sheep and goat.

Comparative FISH-mapping among Y chromosomes of cattle (Bos taurus, 2n = 60, BTA, submetacentric Y chromosome), zebu (Bos indicus, 2n = 60, BIN, acrocentric Y chromosome but with visible small p-arms), river buffalo (Bubalus bubalis, 2n = 50, BBU, acrocentric Y chromosome), sheep (Ovis aries, 2n = 54, OAR, small metacentric Y chromosome) and goat (Capra hircus, 2n = 60, CHI, Y-chromosome as in sheep) was performed to extend the existing cytogenetic maps and improve the understanding of karyotype evolution of these small chromosomes in bovids. C- and R-banding comparison were also performed and both bovine and caprine BAC clones containing the SRY, ZFY, UMN0504, UMN0301, UMN0304 and DYZ10 loci in cattle and DXYS3 and SLC25A6 in goat were hybridized on R-banded chromosomes by FISH. The main results were the following: (a) Y-chromosomes of all species show a typical distal positive C-band which seems to be located at the same region of the typical distal R-band positive; (b) the PAR is located at the telomeres but close to both R-band positive and ZFY in all species; (c) ZFY is located opposite SRYand on different arms of BTA, BIN, OAR/CHI Y chromosomes and distal (but centromeric to ZFY) in BBU-Y; (d) BTA-Y and BIN-Y differ as a result of a centromere transposition or pericentric inversion since they retain the same gene order along their distal chromosome regions and have chromosome arms of different size; (e) BTA-Y and BBU-Y differ in a pericentric inversion with a concomitant loss or gain of heterochromatin; (f) OAR/CHI-Y differs from BBU-Y for a pericentric inversion with a major loss of heterochromatin and from BTA and BIN for a centromere transposition followed by the loss of heterochromatin.

Chromosome fragility in two sheep flocks exposed to dioxins during pasturage.

In the last 3 years several farms raising cattle, river buffalo and sheep have been unable to sell dairy milk due to the presence of high levels of dioxins. Furthermore, several cases of abortion (around 25% of total births) and abnormal foetuses (2.5% of total births) were recorded in two flocks of sheep raised in the province of Naples where a higher level of dioxins (5.27 pg/g fat, as human WHO TCDD equivalent) have been found in the milk mass than that permitted (3.0 pg/g fat, as human WHO TCDD equivalent). Cytogenetic investigations were carried out on 24 sheep (all females), randomly sampled from the two different flocks, one abnormal foetus and 11 female sheep (control) raised approximately 80 km from the area where the two exposed flocks were raised. Frequencies of aneuploid cells, gaps, chromatid breaks, chromosome breaks, fragments and sister chromatid exchange (SCE) were determined. While no differences were observed between the number of aneuploid cells (15% of total cell population) of both exposed animals and controls, significant (P < 0.001) increases in the frequencies of other chromosome abnormalities (mean chromosome abnormality/cell = 0.76 +/- 1.1) and SCEs (mean SCE/cell = 9.4 +/- 3.7) were found in the exposed animals, compared with the control (mean chromosome abnormality/cell = 0.18 +/- 0.4; mean SCE/cell = 7.1 +/- 3.0). Significantly higher values of SCEs (mean SCE/cell = 10.9 +/- 4.4) were also found in the abnormal foetus compared with the control. Chemical analyses on soil, grass and water at two sites where the two flocks were pastured established that doses of dioxins (17 different types) were below the legally permitted limits.

Comparative mapping of twenty-eight bovine loci in sheep (Ovis aries, 2n = 54) and river buffalo (Bubalus bubalis, 2n = 50) by FISH.

Peripheral blood cell cultures were treated for late incorporation of both BrdU and Hoechst-33258 to obtain R-banding pattern preparations. Twenty-eight bovine cosmids from 19 bovine syntenic groups (U), three of which contain type I loci and 25 which contain microsatellite loci and have previously been assigned to cattle chromosomes, were comparatively FISH-mapped to sheep and river buffalo chromosomes according to the standard karyotypes (13 loci for the first time in the latter species). The results enrich the physical maps of both species with information relative to the following loci and to the corresponding syntenic groups: IDVGA35 and IDVGA53 (U6), IDVGA61 and IDVGA84 (U13), JAB10 (U5), IDVGA41 and IDVGA57 (U27), IDVGA87 (U11), IDVGA32 and IDVGA10 (U19), IDVGA49, IDVGA66 and IDVGA68 (U1), ZNF164 (U23), IDVGA74 and IDVGA70 (U9), IDVGA47, IDVGA46 and IDVGA58 (U21), MAP1B (U14), IDVGA79 (U4), CATHL (U12), IDVGA71 (U8), IDVGA59 (U26), IDVGA29 (U29), IDVGA7 (U7), IDVGA82 (X), IDVGA50 (Y). All mapped loci were localized on homoeologous chromosomes and chromosome regions of the two species, confirming the high degree of chromosome homoeologies between the subfamilies Bovinae and Caprinae.

Fatty acid chain elongation synthesis in eel (Anguilla anguilla) liver mitochondria.

The properties of fatty acid chain elongation synthesis have been investigated in liver mitochondria of the European eel (Anguilla anguilla). The incorporation of [1-(14)C]acetyl-CoA into fatty acids shows a specific activity of 0.43+/-0.05 nmol/min x mg protein (n=6), which is more than twice higher than that previously reported in rat liver mitochondria. Label incorporation into fatty acids was, in mitochondria disrupted by freezing and thawing, much higher than in intact organelles thus suggesting a probable localization of this pathway inside mitochondria. Only a negligible acetyl-CoA incorporation into fatty acids occurs in the absence of ATP, Mg2+ or reduced pyridine nucleotides; NADH alone seems to be as effective as NADH + NADPH as a hydrogen donor for the reducing steps. CoASH, without effect up to 10 microM, showed a strong inhibition at higher concentrations. From the ratio of total radioactivity and radioactivity in carboxyl carbon it can be inferred that in eel-liver mitochondria only chain elongation of preexisting fatty acids occurs. A significant fatty acid chain elongation activity is also present when, instead of acetyl-CoA, [2-(14)C]malonyl-CoA is used as a carbon unit donor. Moreover, the synthesized fatty acids were actively incorporated into phopholipids, mainly phosphatidylcholine, phosphatidylethanolamine and sphyngomyelin.

Change in cholesterol levels and in lipid fatty acid composition in safflower oil fed lambs.

The influence of safflower oil supplemented (5%) diet both on cholesterol level and on lipid fatty acid composition was investigated in serum, liver and muscle (Longissimus dorsi) of lambs. The fatty acid pattern varied similarly in all the three tissues. An increase in linoleic acid and in some other polyunsaturated fatty acids, and a simultaneous reduction of the saturated fatty acids (mostly palmitic and stearic acid) were observed. On the contrary, cholesterol content decreased notably (44%) in the liver, with a much less evident decrease in serum and muscle of treated animals.

[Structural analysis of the gene for the beta chain of the antigen-specific receptor of mammalian T-lymphocytes]. / Analisi strutturale dei geni per la catena beta del recettore antigene specifico dei T-linfociti nei mammiferi.

The antigen specific receptor of T cells (TCR) is composed of alpha and beta chains and is normally present on the T cell surface complexed with the components which make up T3. In the case of beta chain, multiple somatic DNA rearrangements bring together V beta (variable), D beta (diversity) and J beta (joining) gene segments before a mature messenger RNA can be transcribed. So far beta chain genes have been extensively studied in the human and in the mouse system and we have very little information on other mammals. Our aims were to obtain information that may provide a structural basis for understanding developmental as well as evolutionary aspects of the TCR gene system in mammals. In this study we compare the hybridization pattern between a human cDNA probe coding for the beta chain constant region and restricted genomic DNA extracted from lymphocytes deriving from human as well as from rat and lamb. The comparison of the hybridization data represent a first piece of information about the variation of the structure of the TCR beta chain genes in mammals.

Cloning and cDNA sequence of the rat X-chromosome linked phosphoglycerate kinase.

This paper reports the isolation and the sequence determination of rat phosphoglycerate kinase (PGK) cDNA clones. This cDNA, derived from an X-linked PGK gene transcript, contains a reading frame of 1254 nt and 5' and 3' non coding regions of 40 and 380 nt respectively. Analysis of the nucleotide sequence at the three codon position shows a biased codon usage with a prevalence of the triplet G non G N. Comparison of the inferred rat amino acid sequence with that of other organisms makes possible the calculation of the unit evolutionary period (UEP) for this enzyme, placing it at around 40 million years (My). Thus PGK is one of the oldest housekeeping enzymes.