Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5 cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen-and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogeninduced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.Mitogens stimulate protein and RNA synthesis (56, 65). The increase in protein synthesis is partly due to increased initiation on preexisting mRNAs, with the result that those mRNAs are recruited into larger polysomes. In addition to an increase in basal translation, specific mRNAs are preferentially upregulated, suggesting that mitogenic signal transduction pathways impinge on the components of the translation machinery that interact with the mRNA. mRNAs are brought to the ribosome by eukaryotic initiation factor (eIF4F) (for reviews, see references 58, 60, and 61). eIF4F is a multiprotein complex formed from 25-, 46-and 220-kDa subunits, called eIF4E, eIF4A, and eIF4G, respectively. eIF4E, also known as cap-binding protein, is responsible for binding the 5Ј-terminal 7-methyl-GTP (m 7 GTP) cap found on all eukaryotic mRNAs. eIF4A is a subunit of an RNA helicase that seems to unwind secondary structure in the mRNA. eIF4G is the scaffolding subunit, to which the other subunits bind. It also has a binding site for eIF3, which links the eIF4F-mRNA complex to the 40S ribosomal subunit. In yeast, eIF4G has an additional functional region, to which the poly(A)-binding protein and the 3Ј end of the mRNA bind (64). Besides serving as a passive scaffold, eIF4G plays a regulatory role, stimulating the binding of capped mRNA to eIF4E (24). Thus, the eIF4F complex promotes interactions between the 5Ј end of the mRNA, the ribosome, and an RNA helicase.As the main mRNA-binding component of the translation machinery, the eIF4F complex has the potential to distinguish between mRNAs for differential translat...

Mammaglobin, a promising diagnostic marker for breast cancer, forms a covalent complex with lipophilin B. mRNA levels for each component of the complex were determined for a number of breast tumors and normal tissues, and correlation of message expression was highly significant between mammaglobin and lipophilin B (p < 0.0001). The complex was purified by both standard biochemical techniques and immunoaffinity chromatography. N-Terminal sequencing revealed that mammaglobin and lipophilin B are processed as predicted by cleavage of their signal sequence after amino acids 19 and 21, respectively. Three molecular masses-representing the fully glycosylated form, the complex without one of the carbohydrate chains, and the deglycosylated proteins-are detected by ProteinChip array SELDI-TOF mass spectrometry after partial enzymatic deglycosylation. This is consistent with the two predicted N-linked glycosylation sites in the primary sequence of mammaglobin and each site having an attached sugar of approximately 3500 Da. Reducing agents release lipophilin B from mammaglobin, and the free peptides are seen at their predicted molecular masses in the deglycosylated complex. Molecular modeling, secondary structure prediction, and circular dichroism indicate that the complex is a small alpha-helical globule that has three disulfide bridges and a carbohydrate chain at each pole. LC-ESI-MS shows that mammaglobin and lipophilin B are bonded in a head to tail orientation. This work describes the biochemistry of the mammaglobin/lipophilin B complex and lays the framework for use of this complex as a novel protein-based serological marker for breast cancer.

In recent years a consensus has emerged from molecular phylogenetic investigations favoring a common endosymbiotic ancestor for all chloroplasts. It is within this conceptual framework that most comparative analyses of eukaryotic biochemistry and genetics now are interpreted. One of the first and most influential sources of data leading to this consensus is the remarkable similarity in genome content among all major plastid lineages. Here we report statistical analyses of two sequence data sets, genes encoding ribosomal proteins and transfer RNAs, from representatives of the three primary plastid lineages and a mitochondrion. The latter almost certainly originated in an independent endosymbiotic association and serves as a control for similarity due to convergent evolution. When genes related to organelle‐specific function are factored out, plastid genomes appear to be no more similar to each other than they are to the mitochondrion. Total similarities in gene content, measured as deviations from the expectation from a process of random gene loss, are correlated with the extent of reduction in the two genomes compared. They do not appear to reflect putative evolutionary relationships among plastids. These analyses indicate that similarities in plastid genome content are better explained by convergent evolution due to constraint on gene loss than by a shared evolutionary history. A review of other data cited as support for a single plastid origin suggests that the alternative hypothesis of multiple origins is at least equally consistent in most cases.

Although nonneurologic organ dysfunction (NNOD) has been shown to significantly affect mortality in subarachnoid hemorrhage, the contribution of NNOD to mortality after severe traumatic brain injury (TBI) has yet to be defined. We hypothesized that NNOD has a significant impact on mortality after severe TBI. The trauma registry was queried for all patients admitted between January 2004 and December 2004 who died during their initial hospitalization after severe TBI (head Abbreviated Injury Score 3 or greater). Cause of death and contributing factors to mortality were determined by an attending trauma surgeon from the medical record. The data were analyzed using both Fisher's exact and Wilcoxon rank sum. One hundred thirty-five patients met inclusion criteria. Sixty-seven per cent were males, 83 per cent were white, and the mean age was 38.5 years. Mean length of stay was 2.9 days. Fifty-four patients (40%) had isolated TBI (chest Abbreviated Injury Score = 0, abdominal Abbreviated Injury Score = 0). Of the 81 deaths attributed to a single cause, 48 (60%) patients died from nonsurvivable TBI or brain death, whereas 33 (40%) died of a nonneurologic cause. Cardiovascular and respiratory dysfunction (excluding pneumonia) contributed to mortality in 51.1 per cent and 34.1 per cent of patients, respectively. NNOD contributes to approximately two-thirds of all deaths after severe TBI. These complications occur early and are seen even among those with isolated head injuries. These findings demonstrate the impact of the extracranial manifestations of severe TBI on overall mortality and highlight potential areas for future intervention and research.