Prospective audit of adverse reactions occurring in 459 primary antibody-deficient patients receiving intravenous immunoglobulin.

Intravenous immunoglobulin (IVIG) is used as the standard replacement therapy for patients with primary antibody deficiencies. A previous study of adverse reactions in patients self-infusing at home over 1 year showed an overall reaction rate of 0.7%. A larger prospective study is reported here, involving a greater number of immunology centres and including children and adults who received infusions from medical or nursing staff as well as those self-infusing. Four hundred and fifty-nine patients were entered into this study and 13 508 infusions were given. The study showed that no severe reactions occurred and the reaction rate was low at 0.8%. This figure could have been lower, 0.5%, if predisposing factors responsible for some reactions had been considered before infusion. In conclusion, the study shows the importance of ongoing training for patients and staff to recognize the predisposing factors to prevent avoidable reactions. Because none of these reactions were graded as severe, the present guidance to prescribe self-injectable adrenaline for patients infusing outside hospital should be reviewed.

Mik1 levels accumulate in S phase and may mediate an intrinsic link between S phase and mitosis.

Two paradigms exist for maintaining order during cell-cycle progression: intrinsic controls, where passage through one part of the cell cycle directly affects the ability to execute another, and checkpoint controls, where external pathways impose order in response to aberrant structures. By studying the mitotic inhibitor Mik1, we have identified evidence for an intrinsic link between unperturbed S phase and mitosis. We propose a model in which S/M linkage can be generated by the production and stabilization of Mik1 protein during S phase. The production of Mik1 during unperturbed S phase is independent of the Rad3- and Cds1-dependent checkpoint controls. In response to perturbed S phase, Rad3-Cds1 checkpoint controls are required to maintain high levels of Mik1, probably indirectly by extending the S phase period, where Mik1 is stable. In addition, we find that Mik1 protein can be moderately induced in response to irradiation of G(2) cells in a Chk1-dependent manner.

A Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins.

The conserved PIK-related kinase Rad3 is required for all DNA-integrity-checkpoint responses in fission yeast. Here we report a stable association between Rad3 and Rad26 in soluble protein extracts. Rad26 shows Rad3-dependent phosphorylation after DNA damage. Unlike phosphorylation of Hus1, Crb2/Rhp9, Cds1 and Chk1, phosphorylation of Rad26 does not require other known checkpoint proteins. Rad26 phosphorylation is the first biochemical marker of Rad3 function, indicating that Rad3-related checkpoint kinases may have a direct role in DNA-damage recognition.

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

UNLABELLED: Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. KEYWORDS: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control.

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.

DNA structure-dependent checkpoints in model systems.

DNA structure dependent checkpoints require a number of proteins which function to arrest the cell cycle in response to DNA damage (such as UV induced lesions) or blocks to DNA replication. Analogous to a signal transduction pathway, checkpoints communicate information between a DNA lesion and the cell cycle machinery. This brief review will focus on yeast model systems which have been instrumental in identifying the various components (initiating signal, detection, signal transduction and cell cycle effector) of the checkpoint pathways. The biological significance of these pathways in mammalian cells is illustrated in patients with ataxia telangiectasia (AT), a multi-system cancer-prone disorder in which DNA damage checkpoints affecting both DNA replication and mitosis are lost. ATM, the gene mutated in this disorder is structurally related to the yeast rad3/MEC1 checkpoint genes. This demonstrates the high degree of evolutionary conservation of checkpoints amongst eukaryotic organisms.

The Schizosaccharomyces pombe rad3 checkpoint gene.

The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution.

The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes.

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia- and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are approximately 300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination.

The chk1 pathway is required to prevent mitosis following cell-cycle arrest at 'start'.

BACKGROUND: The G2-M-phase transition is controlled by cell-cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. Genetic analyses in yeast have defined two related, but distinct, pathways which prevent mitosis--one which acts when S phase is inhibited, and one which acts when the DNA is damaged. In the fission yeast Schizosaccharomyces pombe, many of the gene products involved have been identified. Six 'radiation checkpoint' (rad) gene products are required for both the S-M and DNA-damage checkpoints, whereas Chk1, a putative protein kinase, is required only for the DNA-damage checkpoint and not for the S-M checkpoint following the inhibition of DNA synthesis. RESULTS: We have genetically defined a third mitotic control checkpoint pathway in fission yeast which prevents mitosis when passage through 'start' (the commitment point in G1) is compromized. In cycling cells arrested at start, mitosis is prevented by a Chk1-dependent pathway. In the absence of Chk1, G1 cells attempt an abortive mitosis with a 1C DNA content without entering S phase. Similar results are seen in the absence of Rad17, a typical example of a rad gene product. CONCLUSIONS: Genetic dissection of checkpoints in logarithmically growing fission yeast has identified a pathway that couples mitosis to correct passage through start. This pathway is related to the DNA-structure check-points which ensure that mitosis is dependent on the completion of replication and the integrity of the DNA. We propose that all three mitotic control checkpoints monitor distinct DNA or protein structures at different stages in the cell cycle.

Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.

The tyrosinase gene is expressed specifically in melanocytes and the cells of the retinal pigment epithelium, which together are responsible for skin, hair, and eye color. By using a combination of DNase I footprinting and band shift assays coupled with mutagenesis of specific DNA elements, we examined the requirements for melanocyte-specific expression of the human tyrosinase promoter. We found that as little as 115 bp of the upstream sequence was sufficient to direct tissue-specific expression. This 115-bp stretch contains three positive elements: the M box, a conserved element found in other melanocyte-specific promoters; an Sp1 site; and a highly evolutionarily conserved element located between -14 and +1 comprising an E-box motif and an overlapping octamer element. In addition, two further elements, one positive and one negative, are located between positions -185 and -150 and positions -150 and -115, respectively. We also found that the basic helix-loop-helix factor encoded by the microphthalmia gene, which is essential for melanocyte differentiation, can transactivate the tyrosinase promoter via the M box and the conserved E box located close to the initiator. Since in vitro assays failed to identify any melanocyte-specific DNA-binding activity, the possibility that the specific arrangement of elements within the basal tyrosinase promoter determines melanocyte-specific expression is discussed.

The small heat-shock protein Hsp26 of Saccharomyces cerevisiae assembles into a high molecular weight aggregate.

Hsp26 is one of the major small heat-shock proteins (Hsp) of the yeast Saccharomyces cerevisiae, yet its cellular role remains to be discovered. To examine the cellular consequences of overexpression of Hsp26, the gene encoding this protein (HSP26) was overexpressed from a multicopy plasmid using either its own promoter or by coupling it to the efficient constitutive PGK promoter. The PGK promoter provided the opportunity to overexpress Hsp26 under non-stress conditions and such high level synthesis, prior to a lethal heat shock (50 degrees C), gave a small but reproducible elevation in thermotolerance. In transformed strains overexpressing Hsp26 under either stressed or non-stress conditions, the Hsp26 polypeptide was recovered almost exclusively as a high molecular weight aggregate. This high molecular weight aggregate (or heat-shock granule; HSG) was purified by differential centrifugation and sucrose gradient density centrifugation and shown, by electron microscopic analysis, to be of a uniform size (15-25 nm diameter). Analysis of the purified HSG demonstrated that it had a molecular weight of 550 kDa, yet contained no other integral polypeptides or other macromolecules.

Platelet and coagulation function in patients with abnormal cardiac valves treated with sulphinpyrazone.

Eight patients on warfarin with rheumatic heart disease and prosthetic cardiac valves were selected for study on the basis of persistently elevated plasma beta-thromboglobulin (beta-tg) and platelet factor 4 (PF4) concentrations. Platelet mean lifespan and fibrinogen half life were short, and positively correlated, and both were inversely related to the plasma concentration of the platelet specific proteins. Antithrombin III (ATIII) levels were also reduced. Treatment with sulphinpyrazone resulted in lengthening of both platelet and fibrinogen survival, a rise in ATIII but no change in the beta tg or PF4 concentrations. It is concluded that patients with abnormal cardiac valves and raised plasma levels of beta tg or PF4 have, despite warfarin, a consumption coagulopathy that can be inhibited by sulphinpyrazone.