Chromosome structure deficiencies in MCPH1 syndrome.

Mutations in the MCPH1 gene result in primary microcephaly in combination with a unique cellular phenotype of defective chromosome condensation. MCPH1 patient cells display premature chromosome condensation in G2 phase of the cell cycle and delayed decondensation in early G1 phase, observable as an increased proportion of cells with prophase-like appearance. MCPH1 deficiency thus appears to uncouple the chromosome cycle from the coordinated series of events that take place during mitosis such as some phases of the centrosome cycle and nuclear envelope breakdown. Here, we provide a further characterization of the effects of MCPH1 loss-of-function on chromosome morphology. In comparison to healthy controls, chromosomes of MCPH1 patients are shorter and display a pronounced coiling of their central chromatid axes. In addition, a substantial fraction of metaphase chromosomes shows apparently unresolved chromatids with twisted appearance. The patient chromosomes also showed signs of defective centromeric cohesion, which become more apparent and pronounced after harsh hypotonic conditions. Taking together, the observed alterations indicate additional so far unknown functions of MCPH1 during chromosome shaping and dynamics.

Strategies for chemical cleaning in large scale membrane bioreactors.

Systematically testing alternative cleaning agents and cleaning procedures on a large scale municipal membrane bioreactor, the Erftverband optimized the cleaning strategies and refined the original cleaning procedures for the hollow fiber membranes in use. A time-consuming, intensive ex-situ membrane cleaning twice a year was initially the regular routine. By introducing the effective means of cleaning in place in use today, which employs several acidic and oxidative/alkaline cleaning steps, intensive membrane cleaning could be delayed for years. An overview and an assessment of various cleaning strategies for large scale plants are given.

MCPH1, mutated in primary microcephaly, is required for efficient chromosome alignment during mitosis.

MCPH1 gene, mutated in primary microcephaly, regulates cell progression into mitosis. While this role has been extensively investigated in the context of DNA damage, its function during unperturbed cell cycles has been given less attention. Here we have analyzed the dynamics of chromosome condensation and cell cycle progression in MCPH1 deficient cells under undamaging conditions. Our study demonstrates that chromosome condensation is uncoupled from cell cycle progression when MCPH1 function is lacking, resulting in cells that prematurely condense their chromosomes during mid G2-phase and delay decondensation at the completion of mitosis. However, mitosis onset occurs on schedule in MCPH1 deficient cells. We also revealed active Cdk1 to be mandatory for the premature onset of chromosome condensation during G2 and the maintenance of the condensed state thereafter. Interestingly, a novel cellular phenotype was observed while monitoring cell cycle progression in cells lacking MCPH1 function. Specifically, completion of chromosome alignment at the metaphase plate was significantly delayed. This deficiency reveals that MCPH1 is required for efficient chromosome biorientation during mitosis.

Acyl-CoA dependent acylation of phospholipids in the chloroplast envelope.

Acyl-CoAs are substrates for acyl lipid synthesis in the endoplasmic reticulum. In addition, they may also be substrates for lipid acylation in other membranes. In order to assess whether lipid acylation may have a role in plastid lipid metabolism, we have studied the incorporation of radiolabelled fatty acids from acyl-CoAs into lipids in isolated, intact pea chloroplasts. The labelled lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol and free fatty acids. With oleoyl-CoA, the fatty acid was incorporated preferably into the sn-2 position of PC and the acylation activity mainly occurred in fractions enriched in inner chloroplast envelope. Added lysoPC stimulated the activity. With palmitoyl-CoA, the fatty acid was incorporated primarily into the sn-1 position of PG and the reaction occurred at the surface of the chloroplasts. As chloroplast-synthesized PG generally contains 16C fatty acids in the sn-2 position, we propose that the acylation of PG studied represents activities present in a domain of the endoplasmic reticulum or an endoplasmic reticulum-derived fraction that is associated with chloroplasts and maintains this association during isolation. This domain or fraction contains a discreet population of lipid metabolizing activities, different from that of bulk endoplasmic reticulum, as shown by that with isolated endoplasmic reticulum, acyl-CoAs strongly labelled phosphatidic acid and phosphatidylethanolamine, lipids that were never labelled in the isolated chloroplasts.

First small supernumerary ring chromosome carrying 10q euchromatin in a patient with mild phenotype characterized by molecular cytogenetic techniques and review of the literature.

We report the identification and characterization of the first supernumerary ring chromosome 10 containing a considerable proportion of 10q euchromatin by microdissection and reverse painting in a female patient presenting with short stature. Fluorescence in situ hybridization studies showed that the marker chromosome originates from chromosome 10 and includes the euchromatic bands p11.2 and q11.2. The supernumerary marker chromosome 10 was found in 14% of the peripheral blood lymphocytes analyzed. This constitutional mosaic could be confirmed in oral mucosa cells as a second cell system (16%) by interphase FISH using an alphoid centromeric probe for chromosome 10. Parental karyotypes were normal, uniparental disomy for the normal chromosomes 10 could be excluded by microsatellite analysis. The karyotype of the patient detected in peripheral blood cells can be described as mos 47,XX,+mar.rev ish r(10)(p11.2q11.2)(wcp10+,cep10+)/46,XX.

Prenatal diagnosis and molecular cytogenetic characterization of an unusual complex structural rearrangement in a pregnancy following intracytoplasmic sperm injection (ICSI).

We report on a balanced complex chromosomal aberration detected in a fetus after amniocentesis. The pregnancy was achieved after intracytoplasmic sperm injection. GTG-banding revealed a complex structurally rearranged karyotype with a translocation between chromosomes 5 and 15 and an additional paracentric inversion in the der(15) between bands 5q11.2 and 5q15. Ag-NOR staining showed an interstitial active nuclear organizer region in the der(15). Molecular cytogenetic analyses using whole-chromosome-painting probes, comparative genomic hybridization, and multicolor banding did not point to further structural aberrations or imbalances. Therefore, a complex rearrangement with three breakpoints has occurred, and the karyotype can be described as 46,XX,der(5)t(5;15) (q11.2;p12),der(15)t(5;15)(q11.2;p12)inv(5)(q11.2q15).

TNFalpha reduces glutamate induced intracellular Ca(2+) increase in cultured cortical astrocytes.

The proinflammatory cytokine TNFalpha is locally released during various inflammatory CNS diseases and high cerebrospinal fluid (CSF) titers of TNFalpha were found in meningitis patients. We know from previous studies that TNFalpha also depolarizes astrocytes by reducing their inwardly rectifying K+ currents. We have now investigated the effect of TNFalpha on the glutamate induced intracellular Ca2+ increase in astrocytes, a process which seems to be involved in glial mediated modulation of neuronal synaptic transmisssion. Incubation with TNFalpha (50-1000 U/ml for 60 min) reduces the glutamate induced intracellular Ca2+ increase in astrocytes but not in neurons and this seems to be a phenomenon secondary to the TNFalpha induced depolarization. While other proinflammatory cytokines (interleukin 1beta, IL-2, IL-6) did not interfere with the astrocytic glutamate response, incubation in CSF from septic meningitis patients (CSF-SM) also reduced the glutamate induced intracellular Ca2+ increase. The application of a neutralizing anti-TNFalpha antibody to the CSF-SM prior to cell incubation partially restored the glutamate response. Our data suggest that inflammatory molecules such as TNFalpha impair astrocytes' response to glutamate and this may indirectly affect neuronal synaptic transmission.

Two Missense Mutations in the Primary Autosomal Recessive Microcephaly Gene MCPH1 Disrupt the Function of the Highly Conserved N-Terminal BRCT Domain of Microcephalin.

Primary microcephaly MCPH1 is an extremely rare autosomal recessive disorder associated with congenital microcephaly, mental retardation and a distinctive cellular phenotype of misregulated chromosome condensation. The MCPH1 gene encodes an 835-amino acid protein, microcephalin, which contains 1 N-terminal and 2 C-terminal BRCT (BRCA1 C-terminus) domains. BRCT domains are predominantly found in proteins involved in cell cycle control and DNA repair. Here we describe 1 novel and 1 previously reported MCPH1 missense mutation, p.Trp75Arg and p.Ser72Leu, respectively, in the N-terminal BRCT domain of microcephalin associated with severe congenital microcephaly. Both residues are entirely conserved in the MCPH1 orthologs of all vertebrate species and Drosophila. Proliferating lymphocytes of the patients with p.Trp75Arg and p.Ser72Leu show the unique cellular MCPH1 phenotype of misregulated chromosome condensation, indicating that these missense alterations disrupt the function of the N-terminal BRCT domain of the protein. Interestingly, both residues are strictly conserved in BRCT domains of BRCA1. ClustalW alignments show that the residue p.Ser72 of microcephalin corresponds to p.Ser1715 of the N-terminal BRCT domain of BRCA1, while the microcephalin residue p.Trp75 is analogous to p.Trp1718 in the N-terminal BRCT and to p.Trp1837 in C-terminal BRCT domains of BRCA1. Missense alterations for all 3 corresponding BRCA1 residues were described and are predicted to be deleterious resulting in the destabilization of the BRCA1 protein. Our data on the 2 MCPH1 missense alterations provide further evidence for the functional significance of these residues in BRCT domains.

n-alkylglucosides serve as acceptors for galactosyltransferases from rat liver Golgi vesicles.

n-Alkyl alpha- and beta-D-glucopyranosides with different alkyl chain lengths (Glc-O-CxH2x+1) and n-octyl beta-D-thioglucopyranoside (Glc-S-C8H17) were synthesized, and used as acceptors for galactosyltransferases from rat liver Golgi vesicles. Only the beta-anomers were galactosylated and at constant substrate concentration, the reaction rates reached a maximum for medium alkyl chain lengths (C6, C8 and C10). Apparent Km and Vmax values decreased with increasing alkyl chain length. The reaction products were identified as n-alkyl beta-lactosides by means of thin layer chromatography, fast atom bombardment mass spectrometry and 1H-NMR spectroscopy. Competition experiments showed that UDP-Gal: N-acetylglucosamine beta 1-4-galactosyltransferase (EC 2.4.1.38) and not UDP-Gal: glucosylceramide beta 1-4-galactosyltransferase (lactosylceramide synthase, GalT-2) was responsible for the galactosylation of alkyl glucosides.

Sialylation of n-alkyllactosides, galactosides and glucosides by sialyltransferases from rat liver Golgi vesicles.

n-Alkyl alpha- and beta-lactosides, galactosides and glucosides with different alkyl chain lengths (C2, C8, C14 and C20) were synthesized and used as acceptors for sialyltransferases from rat liver Golgi vesicles. The beta-galactosides, beta-glucosides, and both alpha- and beta-lactosides, were sialylated. Keeping the acceptor concentration constant, sialylation rates reached a maximum for the n-octyl alpha- and beta-lactosides, n-octyl beta-galactoside and n-octyl beta-glucoside, respectively. n-Octyl alpha-galactoside and n-octyl alpha-glucoside were not sialylated. The reaction products were characterized by TLC. With n-octyl lactoside and galactoside as acceptors, two major sialylation products were formed. They could be separated by preparative TLC, and their structures were identified as 2-3 and 2-6 sialylated acceptors, respectively, by a combination of periodate oxidation, NaBD4 reduction, permethylation and subsequent analysis by fast atom bombardment mass spectrometry (FAB-MS). The structure of the single product obtained from n-octyl beta-glucoside was determined to be the 2-6 sialylated glucoside. Competition experiments with n-octyl lactoside and lactosylceramide and ganglioside Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer (GM1) as acceptors for sialyltransferases suggested that SAT-I [NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer (GM3) synthase] was at least in part responsible for the 2-3 sialylation of n-octyl lactoside.

Linoleic acid cytotoxicity to bovine lens epithelial cells: influence of albumin on linoleic acid uptake and cytotoxicity.

The high cytotoxicity of linoleic acid (LA) to cultured bovine lens epithelial cells is correlated with high uptake rates for the fatty acid (FA). Both, LA uptake and LA cytotoxicity strongly increase with the increasing LA-to-albumin molar ratio in the culture medium. Cellular uptake and cytotoxicity of LA can be competitively inhibited with the noncytotoxic palmitic acid. The findings may be of interest in view of the low albumin concentration in aqueous humor, resulting in extremely low buffering capacities for free FAs including LA, oleic acid and other cytotoxic cis-unsaturated free FAs, which are strongly raised in pathological situations like diabetes mellitus.