The role of a small heat-shock protein (Hsp) in the acquisition of thermotolerance in cyanobacteria was investigated. Synechococcus sp. PCC 7942 was transformed with an expression vector carrying the coding sequence of the hspA gene encoding a small heat-shock protein from Synechococcus vulcanus under the control of the tac promoter. The transformant which was shown to constitutively express HspA displayed improved viability compared with the reference strain upon transfer from 30 to 50³C in the light. When the heat shock was given in darkness, the survival rate in the reference strain increased greatly, approaching a level similar to that for the HspA expressing strain after heat shock in the light. Expression of HspA increased thermal resistance of photosystem II (PS II) and protected phycocyanin from heat-induced photobleaching. Our results are indicative of a central role for HspA in amelioration of the harmful effect of light during heat stress and identified the possible sites of action of the small Hsp in vivo to be the PS II complex and the light-harvesting phycobilisomes. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Decreased levels of factor VIII (FVIII) limit the amount of thrombin generated at the site of injury, but not the rate that thrombin is neutralised by antithrombin (AT). We hypothesised that FVIII-deficient mice with heterozygous AT deficiency will demonstrate increased thrombin generation and therefore less in vivo bleeding compared to FVIII-deficient mice with normal AT levels. Therefore, we performed tail bleeding experiments in wild-type (WT), heterozygous AT deficient (AT(+/-)) mice, FVIII-deficient (FVIII(-/-)) mice, and FVIII-deficient mice with heterozygous AT deficiency (FVIII(-/-)/AT(+/-)). Amount of bleeding was assessed by measuring absorbance of haemoglobin released from lysed red blood cells collected after tail transection. In addition, we measured thrombin generation, activated partial thromboplastin time (aPTT), and AT activity in plasma from the different mice groups. Tail bleeding was significantly reduced in FVIII(-/-)/AT(+/-) mice compared to FVIII(-/-) mice. On the other hand, there was no difference in tail bleeding between AT(+/-) and wild-type mice. Thrombin generation was dependent on the mice genotype, and increased in the following order: FVIII(-/-) < FVIII(-/-)/AT(+/-) < WT < AT(+/-). The aPTT was not influenced by reduced AT activity (i.e. AT(+/-) genotype), but was significantly prolonged in FVIII(-/-) and FVIII(-/-)/AT(+/-) mice. Using FVIII-deficient mice as an in vivo murine model of reduced thrombin generation, we demonstrated that moderately reduced AT levels increase thrombin generation and decrease bleeding after traumatic tail vessel injury. In agreement with congenital thrombotic conditions, our data elucidate that bleeding phenotypes can be modulated by the balance between procoagulant and anticoagulant proteins.

Summary. Background: Intron 22 inversion (Inv22) of the coagulation factor (F)VIII gene (F8) is a frequent cause of severe hemophilia A. In addition to Inv22, a variety of F8 mutations (1492 unique mutations) causing hemophilia A have been reported, of which 171 involve deletions of over 50 bp (HAMSTeRs database; http://hadb.org.uk/). However, only 10% of these large deletions have been fully characterized at the nucleotide level. Patients and methods: We investigated gene abnormalities in three unrelated severe hemophilia A patients with high titer FVIII inhibitors. They had previously been shown to carry large deletions of the F8, but the precise gene abnormalities remain to be elucidated. Results: Inverse shifting-PCR (IS-PCR) Inv22 diagnostic tests revealed that these patients carried either type I or II Inv22. However, they showed a wild-type (WT) pattern in the IS-PCR Inv22 complementary tests. We further analyzed their X chromosomes to account for the puzzling results, and found that they had different centromeric breakpoints in the Inv22 X chromosomes, adjacent to the palindromic regions containing int22h-2 or -3, and their spacer region, respectively. The connections appeared to be shifted towards the telomere of the WT F8 Xq28, resulting in a new telomere with an additional intact int22h copy. Conclusions: These gene rearrangements might result from double-strand breaks in the most distal regions of the long arms of the Inv22 X chromosomes, followed by DNA restorations using the WT F8 Xq28 by non-homologous end joining or break-induced replication; thus leading to large F8 deletions in severe hemophilia A patients.

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