Beyond building better brains: bridging the docosahexaenoic acid (DHA) gap of prematurity.

Long-chain polyunsaturated fatty acids (LCPUFA) including docosahexaenoic acid (DHA) are essential for normal vision and neurodevelopment. DHA accretion in utero occurs primarily in the last trimester of pregnancy to support rapid growth and brain development. Premature infants, born before this process is complete, are relatively deficient in this essential fatty acid. Very low birth weight (VLBW) infants remain deficient for a long period of time due to ineffective conversion from precursor fatty acids, lower fat stores and a limited nutritional provision of DHA after birth. In addition to long-term visual and neurodevelopmental risks, VLBW infants have significant morbidity and mortality from diseases specific to premature birth, including bronchopulmonary dysplasia, necrotizing enterocolitis, and retinopathy of prematurity. There is increasing evidence that DHA has protective benefits against these disease states. The aim of this article is to identify the unique needs of premature infants, review the current recommendations for LCPUFA provision in infants and discuss the caveats and innovative new ways to overcome the DHA deficiency through postnatal supplementation, with the long-term goal of improving morbidity and mortality in this at-risk population.

Long-chain polyunsaturated fatty acid levels in US donor human milk: meeting the needs of premature infants?

OBJECTIVE: To determine fatty acid levels in the US donor milk supply. STUDY DESIGN: Donor human milk samples from Iowa (n=62), Texas (n=5), North Carolina (n=5) and California (n=5) were analyzed by gas chromatography. Levels in the Iowa donor milk were compared before and after pasteurization using Student's t-test. Docosahexaenoic acid (DHA) and arachidonic acid (ARA) levels were compared among all milk banks using analysis of variance. RESULT: ARA (0.4 pre, 0.4 post, P=0.18) and DHA (0.073 pre, 0.073 post, P=0.84) were not affected by pasteurization. DHA varied between banks (P<0.0001), whereas ARA did not (P=0.3). DHA levels from all banks were lower than published values for maternal milk and infant formula (P<0.0001). CONCLUSION: Pasteurization of breastmilk does not affect DHA or ARA levels. However, DHA content in US donor milk varies with bank location and may not meet the recommended provision for preterm infants.

The expression of Ki-67, MCM3, and p27 defines distinct subsets of proliferating, resting, and differentiated cells.

The mini-chromosome maintenance proteins (MCM), which are involved in the control of DNA replication, and the cyclin-dependent kinase inhibitors, such as p27/KIP1, represent two groups of proteins that are currently under investigation as diagnostic tumour markers. The expression of p27 and MCM3 was compared with the expression of the Ki-67 protein, an approved marker for proliferating cells, extensively used in histopathology and cancer research. The expression pattern of all three proteins was assessed on germinal centres and oral mucosa, which display a well-defined spatio-temporal organization. The expression of the p27 protein was closely related to differentiated cells, whereas MCM3 and Ki-67 were predominantly localized to the regions of proliferating cells. However, it is important to note that considerable numbers of cells that were growth-arrested, as confirmed by the absence of the Ki-67 protein, stained positive for the MCM3 protein. These results were verified in vitro using growth-arrested Swiss 3T3. The MCM3 protein is therefore expressed in cells that have ceased to proliferate, but are not terminally differentiated, according to the absence of p27 protein expression. In conclusion, a combined analysis of Ki-67, MCM3, and p27 protein expression may provide a more detailed insight into the cell proliferation and differentiation processes that determine individual tumour growth.

Subcellular localization of the human proto-oncogene protein DEK.

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.

The human origin recognition complex protein 1 dissociates from chromatin during S phase in HeLa cells.

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.

Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease.

Mcm (minichromosome maintenance) proteins are important components of the eukaryotic replication initiation apparatus. We investigate the binding of human Mcm proteins to HeLa cell chromatin using micrococcal nuclease as a tool. In previous work we prepared chromatin under low ionic strength conditions. The use of a low salt buffer was necessary to prevent the dissociation of Mcm proteins. Here we use chromatin prepared at more physiological salt concentrations (100 mM NaCl) following the procedure of Fujita et al. (J. Biol. Chem. 272, 10928-10935; 1997) who had shown that ATP stabilizes the interaction of Mcm proteins with chromatin. We show here that micrococcal nuclease released Mcm proteins early during the digestion process suggesting that Mcm proteins reside on chromatin sites which are more open to nuclease attack than bulk chromatin. Released Mcm proteins sedimented through glycerol gradients as a multiprotein complex comprising several of the six known human Mcm proteins.

Human minichromosome maintenance proteins and human origin recognition complex 2 protein on chromatin.

Minichromosome maintenance (Mcm) proteins and the constituents of the origin recognition complex (Orc) are essential components of the eukaryotic replication initiation apparatus. Published evidence strongly suggests that the binding of Mcm proteins to chromatin is contingent upon the prior binding of Orc proteins. Here we use two different approaches to investigate the presence of the human ORC2 protein and of Mcm proteins on chromatin of HeLa cells in various cell cycle phases. First, we mobilized chromatin-bound proteins by micrococcal nuclease and analyzed the resulting digestion products by sucrose gradient centrifugations. Under digestion conditions when Mcm proteins were almost entirely released from chromatin, ORC2 protein was found to be associated with chromatin fragments containing several hundred base pairs of DNA. Second, we used an in vivo cross-linking procedure to covalently link Mcm proteins and ORC2 to DNA by short exposure of intact HeLa cells to formaldehyde. Specific immunoprecipitations revealed that cross-linked nucleoprotein fragments carried either Mcm proteins or ORC2 protein, but not both. Based on the lengths of the DNA fragments in immunoprecipitates, we estimate that the distance between chromatin-bound ORC2 protein and chromatin-bound Mcm proteins must be at least 500-1000 base pairs in HeLa cells.

Human protein MCM6 on HeLa cell chromatin.

Minichromosome maintenance (Mcm) proteins perform essential functions regulating the replication of chromatin. Human cells, like other eukaryotic cells, express at least six Mcm proteins conserved in the central region. We have earlier described the primary structures of five human Mcm proteins, but the primary structure of the sixth human Mcm protein, MCM6, was identified only recently. We now use antibodies, specific for the MCM6 protein, to assess its intranuclear distribution. We find that a fraction of MCM6 protein occurs in the nucleosol, forming multiprotein complexes with other Mcm proteins. More importantly, we use for the first time micrococcal nuclease as a tool to investigate the association of MCM6 protein with chromatin. After short digestion times, a considerable fraction of the MCM6 protein is released from chromatin as a multiprotein complex that includes other Mcm proteins as well. In addition, fractions of MCM3 and MCM6 proteins are released by nuclease digestion as monomeric proteins indicating that at least these two Mcm proteins may also occur as single molecules on chromatin. The data also suggest that the chromatin regions with bound Mcm proteins are more vulnerable to nuclease attack than bulk chromatin and may therefore differ in the arrangement of nucleosomes.

Transcription factor Oct1 binds to the AT-rich segment of the simian virus 40 replication origin.

A cellular protein that binds to the AT-rich late segment of the simian virus 40 (SV40) origin of replication has been identified as transcription factor Oct1. This conclusion is based on the following observations: the late origin binding protein has a molecular mass of about 100 kDa, like factor Oct1, and shares other biochemical properties with Oct1; its binding to the origin is inhibited by antibodies directed against the POU domain of factor Oct1; the isolated POU domain of Oct1 specifically binds to the SV40 late origin region. Thus, the SV40 genome contains binding sites for transcription factor Oct1 in the origin of replication in addition to the previously characterized octamer sites in the viral promoter enhancer. Oct1, bound to the viral origin, negatively affects the DNA unwinding reaction catalyzed by the viral replication initiator T antigen, suggesting that Oct1 may have a role in the regulation of viral replication.

The human EPRS locus (formerly the QARS locus): a gene encoding a class I and a class II aminoacyl-tRNA synthetase.

Glutamyl-tRNA synthetase and prolyl-tRNA synthetase belong to different classes of aminoacyl-tRNA synthetases that are thought to have evolved along independent evolutionary pathways. However, both enzymes are on one polypeptide chain encoded by a single human gene, the EPRS locus, which is transcribed as one long mRNA. We report the structure of the human EPRS gene, which consists of 29 exons spread over at least 90 kb of genomic DNA. The exons, encoding the glutamyl-specific and the prolyl-specific parts of the enzyme, are each clustered in 10-kb sections located at opposite ends of the gene. These two exon clusters are separated by a long intervening DNA section with a number of exons, encoding functions that may be involved in the organization of the mammalian multienzyme synthetase complex. The upstream gene region shows structural features of a regulated gene, and preliminary experiments suggest that the gene is expressed at specific times in growth-stimulated cultured cells. We have localized the gene to the distal long arm of human chromosome 1 and to a corresponding site in mouse chromosome 1.

Effects of the cellular p53 protein on Simian-virus-40-T-antigen-catalyzed DNA unwinding in vitro.

It is known that large T antigen, the regulatory protein encoded by Simian virus 40 (SV40), forms tight complexes with the cellular p53 protein in SV40-transformed rodent cells. Using immunoaffinity procedures we have purified large T antigen and, in separate experiments, the cellular p53 protein. The two proteins formed complexes in vitro which bound well to double-stranded DNA fragments although in a sequence-unspecific manner. Free, uncomplexed T antigen readily converted double-stranded DNA into a single-stranded form whereas in-vitro-formed p53-T-antigen complexes were inactive in this reaction. We conclude that one function of p53 in SV40-transformed mouse cells could be the inhibition of the replication initiating activity of T antigen.

Analysis of a large-T-antigen variant expressed in simian virus 40-transformed mouse cell line mKS-A.

Earlier reports had suggested that the large T antigen expressed in simian virus 40 (SV40)-transformed mKS-A cells may be replication defective. Our experiments support these earlier observations showing that the mKS-A T antigen has a reduced DNA-unwinding activity in vitro. To investigate the molecular basis for this defect, we have isolated from an mKS-A genomic library an EMBL-3 bacteriophage clone carrying in its insert a full-length SV40 DNA element that most likely encodes the expressed T-antigen variant. DNA sequencing revealed only one nonconservative amino acid exchange, Asp to Asn at residue 636. Surprisingly, when a plasmid clone carrying the mKS-A T-antigen-coding sequence was transfected into monkey cells, we found that it replicated quite efficiently, probably suggesting that a high nuclear concentration of the variant T-antigen form compensates for the partial biochemical defect. However, a high nuclear concentration of T antigen was also found in mKS-A T-antigen-transformed mouse cells, yet a fusion of these cells to permissive monkey cells failed to induce in situ replication and excision of integrated SV40 DNA. We discuss possible reasons for the different behavior of T antigen in monkey cells and in mouse cells and suggest that one possibility for the replication-negative phenotype in transformed cells may be related to the fact that T antigen forms a tight complex with the cellular p53 protein in mouse cells but not in monkey cells.

High-affinity SV40 T-antigen binding sites in the human genome.

We describe two different approaches to isolate human genomic sequences possessing high-affinity binding sites for the simian virus 40 (SV40) large T antigen. First, SV40 T antigen was added to Sau3A-restricted human DNA; the resulting T-antigen-DNA complexes were collected after repeated passages through nitrocellulose filters. The second approach involves the specific immunoprecipitation of chromatin fragments, generated by Sau3A treatment of nuclear chromatin from SV40-transformed human cells. The DNA fragments obtained were cloned in plasmid vectors for further investigation. Using the filter binding approach we isolated four different fragments with high-affinity binding sites. The binding site in one fragment was related to the strong T-antigen binding site I in the SV40 genome. The other three fragments contained multiple recognition pentamers, GA(G)GGC. Only one fragment with a high-affinity binding site was identified among the immunoprecipitable chromatin fragments. This DNA fragment belongs to the L1 family of human repetitive DNA. We present evidence suggesting that a significant fraction of human L1 elements possesses T-antigen binding sites. L1-related sequences appear as extrachromosomal elements in an SV40-transformed human cell line, and the amount of extrachromosomal L1 DNA was found to increase after fusion of transformed cells to permissive monkey cells.

DNA sequence of the leftward junction in the adenovirus-simian virus 40 hybrid Ad2+D2 and determination of the structure of the D2-T antigen.

The nucleotide sequence of the junction between the simian virus 40 early region and the adenovirus type 2 late region L4 in the hybrid virus Ad2+D2 was determined. The deduced amino acid sequence suggests that the D2-T antigen is a chimeric protein sharing 594 amino acids with the C-terminal end of the simian virus 40 T antigen and 104 amino acids with the N terminus of the adenovirus type 2 33,000-molecular-weight protein. The predicted structure of the D2-T antigen was confirmed by an immunoprecipitation analysis.