siRNA-mediated AML1/MTG8 depletion affects differentiation and proliferation-associated gene expression in t(8;21)-positive cell lines and primary AML blasts.

The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.

The role of mesopores in intracrystalline transport in USY zeolite: PFG NMR diffusion study on various length scales.

PFG NMR has been applied to study intracrystalline diffusion in USY zeolite as well as in the parent ammonium-ion exchanged zeolite Y used to produce the USY by zeolite steaming. The diffusion studies have been performed for a broad range of molecular displacements and with two different types of probe molecules (n-octane and 1,3,5-triisopropylbenzene) having critical molecular diameters smaller and larger than the openings of the zeolite micropores. Our experimental data unambiguously show that, in contrast to what is usually assumed in the literature, the intracrystalline mesopores do not significantly affect intracrystalline diffusion in USY. This result indicates that the intracrystalline mesopores of USY zeolite do not form a connected network, which would allow diffusion through crystals only via mesopores.

Pulsed-field gradient nuclear magnetic resonance study of transport properties of fluid catalytic cracking catalysts.

Pulsed-field gradient nuclear magnetic resonance (PFG NMR) has been applied to study molecular diffusion in industrial fluid catalytic cracking (FCC) catalysts and in USY zeolite for a broad range of molecular displacements and temperatures. The results of this study have been used to elucidate the relevance of molecular transport on various displacements for the rate of molecular exchange between catalyst particles and their surroundings. It turned out that this rate, which may determine the overall rate and selectivity of FCC process, is primarily related to the diffusion mode associated with displacements larger than the size of zeolite crystals located in the particles but smaller than the size of the particles. This conclusion has been confirmed by comparative studies of the catalytic performance of different FCC catalysts.

CD8beta/CD28 expression defines functionally distinct populations of peripheral blood T lymphocytes.

Peripheral blood CD8+ T lymphocytes generally express the CD8 coreceptor as an alphabeta heterodimer. On these cells, the CD8beta chain is present either at high (CD8betahigh) or low density (CD8betalow). CD8betahigh cells are CD28+, whereas CD8betalow cells are CD28+ or CD28-. Therefore, three subpopulations of CD8+ T cells can be described: (i) CD8betahighCD28+ (ii) CD8betalowCD28+, and (iii) CD8betalowCD28- cells. Phenotypic and functional characterization of these CD8+ T cell subsets revealed significant differences. CD8betahighCD28+ cells predominantly express CD45RA. In contrast, CD8betalowCD28+ cells frequently express CD45R0 and the activating NK receptor CD161. CD8betalowCD28- cells frequently revert to the CD45RA phenotype. In addition, these cells express CD16, CD56, CD94, and the killer-inhibitory receptors NKB1 and CD158a. Intracellular IL-2 was frequently detected in CD8betahighCD28+ cells and CD8betalowCD28+ cells, but not CD8betalowCD28- cells. CD8betalowCD28+ cells and CD8betalowCD28- cells frequently stained positive for IFN-gamma. In addition, these cells contain intracellular perforin and granzyme A. Expression of Fas (CD95) as well as susceptibility to apoptosis is markedly increased in CD8betalowCD28+ and CD8betalowCD28- cells as compared to CD8betahighCD28+ cells. In vitro activation of peripheral blood lymphocytes triggered expansion of CD8betahighCD28+ cells as well as a development into CD8betalowCD28+ and CD8betalowCD28- cells. Similarly, activation of CD8betahighCD28+ cord blood cells resulted in the appearance of CD8betalowCD28+ and CD8betalowCD28- cells. These data suggest that CD8betahighCD28+ cells can differentiate into CD8betalowCD28+ and CD8betalowCD28- cells upon TCR stimulation. Therefore, the CD8beta/CD28 subsets in peripheral blood may reflect distinct stages of post-thymic CD8+T cell development.

CD8+ T cell populations in common variable immunodeficiency.

In a subgroup of CVID T cell abnormalities have been reported. Peripheral blood T lymphocytes from patients with common variable immunodeficiency (CVID) and blood donors were examined for expression of CD8alpha, CD8beta and CD28. In CVID, three CD8+ T cells could be defined: CD8beta(high)CD28+ (expressing CD8beta at a high median fluorescence intensity), CD8beta(low)CD28+ and CD8beta(low) CD28- cells. The number of CD8beta(low) cells was markedly increased compared to blood donors. After activation, CD8beta(high)CD28+ cells from cord blood differentiated into CD8beta(low) CD28- cells. Therefore, CD8beta(low) cells are induced by activation even in normal donors, but may reflect inflammatory activity in CVID.

Persistent antihypertensive effect of oral nitrite supplied up to one year via the drinking water in spontaneously hypertensive rats.

The hypothesis was studied whether the chronic administration of nitrite lowers the blood pressure of spontaneously hypertensive rats (SHR) and prevents secondary hypertension-induced organ lesions. For this purpose totally 96 SHR received 50 to 75 mmol/l NaNO2 or equimolar amounts of NaHCO3 in their drinking water during 4, 8 or 12 months. At each point of time arterial blood pressure, determined with the tail cuff method, was significantly lower in the NaNO2-group in comparison to the controls indicating that no significant tolerance towards nitrite had developed. There was also a tendency towards reduced cardiac hypertrophy and renal atrophy in the NaNO2-group, however without reaching the level of significance. Drinking water containing 75 mmol NaNO2/l was not well tolerated by young rats in contrast to 50 mmol/l. Possible beneficial effects of high dietary nitrate/nitrite levels are discussed with respect to the low frequency of hypertension observed in vegetarians.

Molecular characterization of a common fragile site (FRA7H) on human chromosome 7 by the cloning of a simian virus 40 integration site.

Common fragile sites are chromosomal loci prone to breakage and rearrangement, hypothesized to provide targets for foreign DNA integration. We cloned a simian virus 40 integration site and showed by fluorescent in situ hybridization analysis that the integration event had occurred within a common aphidicolin-induced fragile site on human chromosome 7, FRA7H. A region of 161 kb spanning FRA7H was defined and sequenced. Several regions with a potential unusual DNA structure, including high-flexibility, low-stability, and non-B-DNA-forming sequences were identified in this region. We performed a similar analysis on the published FRA3B sequence and the putative partial FRA7G, which also revealed an impressive cluster of regions with high flexibility and low stability. Thus, these unusual DNA characteristics are possibly intrinsic properties of common fragile sites that may affect their replication and condensation as well as organization, and may lead to fragility.

An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness.

The locus for the incomplete form of X-linked congenital stationary night blindness (CSNB2) maps to a 1.1-Mb region in Xp11.23 between markers DXS722 and DXS255. We identified a retina-specific calcium channel alpha1-subunit gene (CACNA1F) in this region, consisting of 48 exons encoding 1966 amino acids and showing high homology to L-type calcium channel alpha1-subunits. Mutation analysis in 13 families with CSNB2 revealed nine different mutations in 10 families, including three nonsense and one frameshift mutation. These data indicate that aberrations in a voltage-gated calcium channel, presumably causing a decrease in neurotransmitter release from photoreceptor presynaptic terminals, are a frequent cause of CSNB2.

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu.

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.

Immunohistochemical pattern of insulin-like growth factor (IGF) I, IGF II and IGF binding proteins 1 to 6 in carcinoma in situ of the testis.

AIM: To study the immunohistochemical localisation of insulin-like growth factor (IGF) I, IGF II, and IGF binding proteins 1-6 in intratubular germ cell neoplasia in the vicinity of solid germ cell tumours of the testis. METHODS: Testes were obtained from 13 patients (20-35 years old) who had undergone orchidectomy for treatment of a solid germ cell tumour. Tumour cells were verified histologically by their distinctive morphology and by visualisation of placental alkaline phosphatase immunoreactivity. RESULTS: The majority of carcinoma in situ (CIS) cells were immunopositive for IGF I, whereas no CIS cells stained for IGF II. Of all the IGF binding proteins investigated, CIS cells showed intense immunoreactivity for IGF binding protein 5 and lower expression of all other IGF binding proteins. CONCLUSIONS: These results suggest that the action of IGF binding protein 5 in CIS cells may modulate the activity of IGF I. This may be related to a proliferative advantage that could facilitate tumour development.

Structural analysis of the murine cell adhesion molecule L1 by electron microscopy and computer-assisted modelling.

In the present study we have analysed the morphology of two fragments with apparent molecular weights of 180 and 140 kDA (L1-180 and L1-140) derived from the extracellular region of the murine neural cell adhesion molecule L1. The fragment L1-180 consists of almost the entire extracellular part of the molecule, and is built up of six immunoglobulin-like and five fibronectin type III-like domains. Fragment L1-140 lacks one-half of the third, the fourth and the fifth fibronectin type III-like domains. By electron microscopic analysis of rotary-shadowed molecules, L1-140 and L1-180 revealed fibrillar structures 31-43 nm long and 7-12 nm wide with one pronounced globular terminal domain. As determined by complex formation with an L1 antibody, this terminal part of the molecule is formed by the fibronectin type III-like domains. The individual structures showed variation and complexity, and four distinct aspects were identified. These different forms probably represent two-dimensional projections of the same three-dimensional helical structure. Computer-assisted modelling of the L1 molecule, i.e. the protein backbone, showed no strong intramolecular interaction between the different fibronectin type III- or Ig-like domains, suggesting that the formation of the globular part of the molecule is probably achieved by protein-carbohydrate and/or carbohydrate-carbohydrates rather than protein-protein interactions. In addition, our model proposes that interactions occur within the interfaces between the different domains. The highly conserved amino acid residues in these regions point to the necessity of maintaining the orientation between the different domains.

[The optimization of group size for fattening rabbits in group housing on grids made of artificial material]. / Untersuchungen zur Optimierung der Gruppengrösse bei Mastkaninchen in Gruppenhaltung auf Kunststoffrosten.

ZIKA-fattening rabbits in groups of 4, 8, 16, 32 and 64 animals (5 rabbits/m2) have been proved in 6 repetitions with all together 144 animals during the fattening period of nine weeks in regard to their fattening performance, health and behaviour. The aim was to find an optimal group size for fattening rabbits with respect to animal welfare. The results show, that fattening performance and health of the rabbits have not been influenced remarkably by group size, whereas behaviour was different in so far, as the rabbits in groups of 16 showed a greater percentage of relaxed positions as well as a remarkable smaller percentage of aggressive behaviour. Therefore the group with 16 fattening rabbits is that, which can be advised for the fattening of rabbits in the Hohenheimer group housing.

The genomic organization of a human creatine transporter (CRTR) gene located in Xq28.

During the course of a large-scale sequencing project in Xq28, a human creatine transporter (CRTR) gene was discovered. The gene is located approximately 36 kb centromeric to ALD. The gene contains 13 exons and spans about 8.5 kb of genomic DNA. Since the creatine transporter has a prominent function in muscular physiology, it is a candidate gene for Barth syndrome and infantile cardiomyopathy mapped to Xq28.

[Scoliosis, lordosis and kyphosis in breeding rabbits]. / Skoliosen, Lordosen und Kyphosen bei Zuchtkaninchen.

Vertebral columns of female and male breeding rabbits, kept in conventional cages or in a group housing system, were investigated by spot checks anatomically and radiographically in regard to deformations of the vertebral column. It should be proved, whether depending on the housing system and the opportunity of locomotion the male and female rabbits get deformations of the vertebral column. The observations show, that the bucks had no deformations, whereas the does had. It became evident, that frequence and degree of deformation were dependent on the cage size. It provokes deformations by "flat sitting" as well as the systemic hypoplasia of bony tissue caused by deficiency of locomotion. Reproduction provokes deformations of the vertebral column, too, causing alterations in the static-dynamic forces of trunk construction as well as a high need and metabolism of calcium. The causing factors and the relevance of animal protection are discussed.

[Testing of various drinking troughs in consideration of the physiological drinking behavior of rabbits]. / Prüfung verschiedener Wassertränken unter Berücksichtigung des physiologischen Trinkverhaltens von Kaninchen.

To investigate the drinking behaviour of rabbits as well as to prove the suitability of different drinker systems under animal suitable conditions all in all 32 rabbits aged four to eight weeks have been held to test in free choice four drinker types usually applied in practice. The rabbits could choose between swimmer-, low pressure-bowl-, nipple- and automatic minidrinkers. In two passages four animals in each of the four boxes have been held for four weeks and watched by video once a week during 24 hours to register the frequency of the animals' drinking at the four alternative drinkers. The choice experiments show that the low pressure bowl-drinker has been preferred in frequency, followed by the nipple drinker. The swimmer-bowl-drinker was less frequented, the automatic minidrinker was avoided in tendency. All in all we see, that by offering bowl- and/or nipple-drinkers we present a natural adequate drinking to the rabbits. Both drinker-systems can be assessed as those with respect to animals' behaviour.

Increase of streptozocin toxicity by magnesium deficiency in the diabetic rat model.

To study interactions between magnesium (Mg) and diabetes mellitus, female SD-rats weighing ca. 230 g were rendered Mg-deficient by offering a diet providing only 20% of the rat's requirement. After 14 days the animals were injected 75 mg streptozocin (STZ) per kg body weight intraperitoneally. Placebo-treated controls received the same diet, however their drinking-water was enriched with 20 mmol/l Mg as the magnesium-L-aspartate hydrochloride. Mg deficiency remarkably increased STZ-induced lethality from 3.8% to 61.1% on day 35. Pronounced hyperglycemia and necrosis of pancreatic beta cells also suggest an increased effect of STZ on the pancreas during Mg deficiency. The underlying mechanisms are discussed. Food consumption was decreased in Mg-deficient animals and steeply increased 7 days following STZ treatment. Similarly consumption of drinking-water also increased. Since diabetic rats lost body weight, relative and absolute Mg intake via food or drinking-water increased. In this way further Mg depletion of diabetic rats was prevented.

Functional topography of the myelin-associated glycoprotein. I. Mapping of domains by electron microscopy.

The functional topography of the myelin-associated glycoprotein (MAG) was investigated by electron microscopic analysis of rotary-shadowed molecules of a MAG fragment (MAG 90) comprising the five immunoglobulin-like domains of the extracellular part of the molecule. MAG 90 molecules appeared as rod-like structures (18.5 +/- 1.2 nm long and 4.0 +/- 0.8 nm wide) with a globular domain at one end. Antibodies directed against the amino- and carboxy-terminus of MAG 90 interacted with the non-globular terminal region, indicating that the molecule is bent in the globular region with the amino- and carboxy-terminal arms in close apposition to each other. An antibody which interferes with the binding of MAG to neurons interacted predominantly with the globular domain of MAG 90. The fibril-forming collagen types I, III and V bound mainly to the non-globular terminal region of MAG 90, whereas the majority of heparin molecules interacted with the globular region of the molecule. The L2/HNK-1 carbohydrate structure was localized at the non-globular region in the protein fragment comprising the fourth and fifth immunoglobulin-like domains.