Molecular oxygen dependent steps in fatty acid oxidation by cyclooxygenase-1

The mechanism by which cyclooxygenase-1 (COX-1), a heme- and tyrosyl radical-containing enzyme, catalyzes the regio- and stereospecific oxygenation of polyunsaturated fatty acids to prostaglandin or hydroperoxide products has not been understood. Steady-state kinetic studies conducted with the native substrate arachidonic acid and the slower substrate linoleic acid are described here. Second-order rate constants, kcat/KM for fatty acid and O2, are found to depend upon the concentration of the other cosubstrate. Competitive oxygen kinetic isotope effects (18O KIEs) kcat/KM(16,16O2)/kcat/KM(18,16O2) reveal that a peroxyl radical is formed in or before the first kinetically irreversible step. Together, the results indicate that the oxygenase reaction occurs by a sequential mechanism which most likely involves reversible abstraction of a hydrogen atom from the fatty acid prior to the trapping of the delocalized substrate radical by O2. The identity of the first kinetically irreversible step, subsequent to forming the peroxyl radical, is also discussed in the context of the magnitude of the oxygen kinetic isotope effects as well as the behavior of kcat/KM(O2) in response to changing solvent pH, pD, and viscosity.     

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.      

Terrigenous sediment-dominated reef platform infilling: an unexpected precursor to reef island formation and a test of the reef platform size–island age model in the Pacific

Low-lying coral reef islands are considered highly vulnerable to climate change, necessitating an improved understanding of when and why they form, and how the timing of formation varies within and among regions. Several testable models have been proposed that explain inter-regional variability as a function of sea-level history and, more recently, a reef platform size model has been proposed from the Maldives (central Indian Ocean) to explain intra-regional (intra-atoll) variability. Here we present chronostratigraphic data from Pipon Island, northern Great Barrier Reef (GBR), enabling us to test the applicability of existing regional island evolution models, and the platform size control hypothesis in a Pacific context. We show that reef platform infilling occurred rapidly (~4–5 mm yr⁻¹) under a “bucket-fill” type scenario. Unusually, this infilling was dominated by terrigenous sedimentation, with platform filling and subsequent reef flat formation complete by ~5000 calibrated years BP (cal BP). Reef flat exposure as sea levels slowly fell post highstand facilitated a shift towards intertidal and subaerial-dominated sedimentation. Our data suggest, however, a lag of ~1500 yr before island initiation (at ~3200 cal BP), i.e. later than that reported from smaller and more evolutionarily mature reef platforms in the region. Our data thus support: (1) the hypothesis that platform size acts to influence the timing of platform filling and subsequent island development at intra-regional scales; and (2) the hypothesis that the low wooded islands of the northern GBR conform to a model of island formation above an elevated reef flat under falling sea levels.

     

Managing the centrosome numbers game: from chaos to stability in cancer cell division

Aneuploid tumor cells can arise through multipolar mitosis caused by supernumerary centrosomes. Multipolar spindles, however, are antagonistic to cell viability. Thus, most cells derived from such an aberrant mitosis would be eliminated by apoptosis. A rare daughter cell, through chance acquisition of an appropriate chromosome complement and/or gene dosage, could survive and contribute to a clone of aneuploid tumor cells. Survival and perpetuation of the clone, however, requires an additional step - the resumption of mitotic stability through the assembly of a bipolar, not multipolar, spindle. Either selective inactivation of the extra centrosomes or their coalescence into two functional spindle poles corrects the problem of centrosome excess. Current data support coalescence as a mechanism for regulating the number of functional centrosomes in tumor cells.     

Romancing mitosis and the mitotic apparatus

One of the earliest lessons students learn in biology is the process of mitosis and how cells divide to produce daughter cells. Although first described more than a century ago by early investigators such as E. B. Wilson, many aspects of mitosis and cell division remain the subject of considerable research today. My personal investigations and research contributions to the study of mitosis were made possible by recent developments in the field when I began my career, including access to novel mammalian cell culture models and electron and fluorescence microscopy. Building upon those innovations, my laboratory and other contemporary investigators first charted the ultrastructure and molecular organization of mitosis and chromosome movement and the assembly and function of the cytoskeleton. This field of research remains a significant challenge for future investigators in cell biology and medicine.     

Dynamics of Human Replication Protein A Subunit Distribution and Partitioning in the Cell Cycle

Human replication protein A (RPA) is a three-subunit protein complex involved in DNA replication, repair, and recombination. To gain insight into the dynamics of subunit assembly, we examined the subcellular distribution of RPA subunits (p70, p34, and p11) during the cell cycle. All three subunits colocalized in G1 and S phases, showing a diffuse nuclear distribution in G1 but a dot-like nuclear pattern in S phase. During S phase, the subunits showed a pattern reminiscent of the replication granules/factories described by others as sites of replication machinery. In metaphase, p70 preferentially associated with the spindle poles, p34 was found on chromosomes, and p11 remained in the cytoplasm. In telophase, p70 and p34 appeared in the forming daughter nuclei; p11 remained in the cytoplasm until G1. Among the three subunits only p34 was associated with the nuclear matrix and this association persisted throughout the cell cycle. We conclude that (i) RPA complex assembly is differentially regulated, (ii) the replication machinery may be anchored to the nuclear matrix, and (iii) RPA subunits partition during mitosis and sort into daughter nuclei by different routes.     

Postoperative radiotherapy and late mortality: evidence from the Cancer Research Campaign trial for early breast cancer.

OBJECTIVE--To identify any excess mortality caused by adjuvant radiotherapy for early breast cancer. DESIGN--Prospective randomised clinical trial. Two thousand subjects needed for study to have a 90% chance of detecting a difference in survival rate of 7% with 95% significance. Patients were followed up until June 1988, giving follow up of 158-216 months. SETTING--A multicentre trial mainly drawing patients from centres in the United Kingdom. PATIENTS--2800 Women presenting with clinical stage I or II carcinoma of the breast from June 1970 to April 1975. INTERVENTIONS--One group of women (n = 1376) had simple mastectomy followed by immediate postoperative radiotherapy (1320 to 1510 rets). The remaining women (n = 1424) had simple mastectomy with subsequent careful observation of the axilla, radiotherapy being delayed until there was obvious progression or recurrence of disease locally. END POINT--Increased mortality in patients treated with radiotherapy from causes other than breast cancer. MEASUREMENTS AND MAIN RESULTS--Survival was measured from time of first treatment to death or last follow up. Deaths from any cause and from specified causes were counted as events. Comparison over the whole follow up showed a slight excess mortality in the group treated with radiotherapy (relative risk 1.04; 95% confidence interval 0.94 to 1.15). The relative risk of death from breast cancer was 0.97 (0.87 to 1.08) but that of death from other causes was 1.37 (1.09 to 1.72), the increase mainly being in women who had had tumours of the left breast (1.61 (1.17 to 2.24)) and had been treated with orthovoltage (1.85 (1.27 to 2.71)). Analysis of causes of death after five years showed a relative risk of 2.11 (1.25 to 3.59) for new malignancies and of 1.65 (1.05 to 2.58) for cardiac disease, the increase in cardiac mortality being most pronounced in patients who had had tumours of the left breast and whose treatment had included orthovoltage radiation (relative risk 2.67 (1.28 to 5.55)). CONCLUSIONS--Adjuvant radiotherapy after simple mastectomy for early breast cancer produces a small excess late mortality from other cancers and cardiac disease. The risk has to be balanced against the higher risk of local recurrence when immediate postoperative radiotherapy is not given. The balance has to be assessed for each patient, and for many patients radiotherapy will still be desirable in the initial treatment of their early breast cancer.