Calcium blockade reduces renal apoptosis during ischemia reperfusion.

Apoptosis is well described in invertebrates and recently documented in mammals. The prevalence and pathophysiology of mammalian apoptosis is unknown and may have clinical ramifications. The aim of this study is to investigate the apoptotic response during kidney ischemia-reperfusion (I/R) injury. Kidney I/R was initiated in anesthetized rats by occlusion of the renal pedicle for 45 min with or without pretreatment with .2 mg/kg verapamil: control animals received sham exposure. Flow was re-established after ischemia and the animals were allowed to recover for 24 h. Bilateral kidneys were harvested for DNA electrophoresis, Western analysis for p53, Northern analysis for c-myc expression, and light and electron microscopic analysis. Kidney I/R caused characteristic DNA laddering in the clamped kidney, and less extensive laddering was seen in the contralateral kidney. Light and electron microscopic analysis confirmed apoptotic morphology in the reperfused tissues. Verapamil pretreatment completely abolished DNA laddering and attenuated the microscopic evidence of apoptosis. p53 levels were increased by I/R in the ischemic kidney and moderately increased in the contralateral organ. c-myc mRNA levels were increased by the I/R insult. Kidney I/R injury may induce global apoptosis, which seems to be associated with an alteration in calcium homeostasis. The increase in p53 and c-myc mRNA levels seen with I/R may facilitate apoptosis. Calcium modulation seems to reduce apoptosis during I/R and may have therapeutic implications.

Electron spin resonance and fluorescence studies of the conformational environment of the thiol groups of thrombospondin: interactions with thrombin.

The free thiols of platelet thrombospondin (TSP) were modified with thiol-specific spin labels and fluorescence probes. The conformational effects of thrombin complexation with TSP were monitored by thiol-specific spin labels covalently attached to TSP and active site specific spin labels on thrombin. The results provide evidence supporting speculations that the thiols of the three polypeptide chains in TSP are not conformationally identical. Studies on the effects of Ca2+ and temperature confirm that TSP exists in multiple conformations which are under dynamic equilibrium. The ESR spectra of spin-labeled TSP are sensitive to the proteolytic effects of thrombin in the presence and absence of calcium. Phenylsulfonyl fluoride spin labels specific for the active site of thrombin are excellent indicators of thrombin: TSP complex formation in the absence of calcium. The anticoagulant thrombin inhibitor hirudin competes with TSP for the same binding locus on thrombin (which includes the requirement of an intact anion exosite). The results suggest that the species observed here is the noncovalent complex formed during the first step of the TSP--thrombin interaction, showing also that thrombin activity is not essential for complex formation. ESR and fluorescence studies of thiol-labeled TSP indicate that the sulfhydryls are not affected in the noncovalent thrombin: TSP complex, although they must be playing a major role in the second step, i.e., formation of the covalent complex, through intermolecular thiol exchange.

Purification and characterization of recombinant polymeric hemoglobin P1 of Glycera dibranchiata.

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the alpha-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).

Localization of two binding domains for thrombospondin within fibronectin.

Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.

The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata.

The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 199-216) showed that all five globins have identical residues at 39 positions. At 44 positions, the three polymeric globins share identical residues that differ from the identical residues at the corresponding locations in the monomeric sequences M-II and M-IV including position E7, where the latter have leucine instead of the distal histidine. At 15 positions, there occurs an alteration from polar to nonpolar or from a small nonpolar to a larger nonpolar residue in going from the monomeric to the polymeric globins.(ABSTRACT TRUNCATED AT 400 WORDS)

The heterogeneity of the polymeric intracellular hemoglobin of Glycera dibranchiata and the cDNA-derived amino acid sequence of one component.

The erythrocytes of the marine polychaete Glycera dibranchiata contain a number of different, single-chain hemoglobins, some of which self-associate into a 'polymeric' fraction. An oligodeoxynucleotide probe was synthesized based on partial amino acid sequences determined by chemical methods, and used to screen a cDNA library constructed from the poly(A+)mRNA of Glycera erythrocytes (Simons, P.C. and Satterlee, J.D. (1989) Biochemistry 28, 8525-8530). The longest positive inserts found were sequenced using the dideoxy nucleotide chain termination method. One complete clone was obtained: clone 5A, 816 bases long, contained 59 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for 147 amino acids and a 3'-untranslated region of 316 bases. The derived amino acid sequence of Glycera globin P1 was in agreement with the partial amino acid sequences obtained by chemical methods. Three additional inserts obtained in the screening were also sequenced: the inferred amino acid sequences proved to be partial globin sequences which were different from each other and from the sequence of P1. Thus, the 'polymeric' fraction of the intracellular hemoglobin of Glycera probably consists of at least four different globin chains much like the 'monomeric' fraction. Comparison of the 'polymeric' sequence with the two known 'monomeric' sequences, M-II and M-IV, shows that they share 54 identical residues. At 74 positions, the identical residues in M-II and M-IV differ from the corresponding residue in P1, including at E-7, where P1 has a distal His, in contrast to Leu in M-II and M-IV. The alignment of Bashford et al. ((1987) J. Mol. Biol. 196, 199-216) and their templates were used to examine the principal differences between the two types of Glycera globin sequences. They appear to consist of uncommon surface amino acid residues at positions C6 (Phe vs. Ala), E10 (Val vs. Lys), E17 (Lys vs. Val), G1 (Arg vs. Lys), G10 (Met vs. Ala) and H5 (Arg vs. Lys). One or more of these residues could be responsible for the self-association exhibited by the 'polymeric' Glycera globins.

Platelet membrane glycoprotein V: characterization of the thrombin-sensitive glycoprotein from human platelets.

Human platelets contain a single membrane glycoprotein which is susceptible to thrombin proteolysis, glycoprotein V. We have purified 1 mg of glycoprotein V from 10(13) platelets using a combination of gel filtration, hydroxylapatite and ion-exchange chromatographies. Glycoprotein V has a blocked amino-terminus. Following proteolysis by human alpha-thrombin, a major fragment, termed glycoprotein Vf1, had the sequence Gly-Pro-Phe-X-Arg-Pro-Ala-Ala-Asp-Glu-Ser-Val-Glu-Ala-Pro-Val-Asn-Gln-Al a-Glu- Ala-Pro-. The purified glycoprotein was not a substrate for human gamma-thrombin. Glycoprotein V contained 17.5% carbohydrate, with the majority of the carbohydrate consisting of neutral hexoses. Deglycosylated glycoprotein V had a molecular weight of 57.5 kDa compared to the glycosylated protein's 82 kDa and the deglycosylated protein was recognized by polyclonal antibodies raised against glycoprotein V. Immunoelectrophoresis of human and rat platelets and megakaryocytes gave a single immunoreactive band, with the rat glycoprotein having a slightly larger molecular mass. Glycoprotein V is most likely an integral membrane protein.

Transcriptional activity at the 3' end of the actin gene at 5C on the X chromosome of Drosophila melanogaster.

Polyribosomal poly(A)+ RNA from stage-specific embryos of Drosophila melanogaster was analyzed by Northern hybridizations to determine the transcription at the 5C actin gene (act5C) and to establish whether these mRNAs are used in translation. A 3' end probe, which contains 30 nucleotides of the actin coding sequence as well as 3'-end-adjacent sequence, hybridizes in addition to the act5C transcript to a smaller transcript 450 nucleotides long. Hybridizations with the 3' end strand-specific probes show that this transcript has the same orientation as the actin gene. This transcript is temporally coexpressed with the act5C gene over most of the developmental stages examined. S1 nuclease mapping and primer extension experiments place its 5' end about 20 nucleotides upstream from the actin translation termination signal. Furthermore, the transcript appears to translate in an in vitro translation system into a protein of 7.4 kDa, as expected from the size of the open reading frame. The data suggest, but do not prove, that the 3'-end-transcribed but untranslated region of act5C may be involved in actin gene expression.

Homology between actin coding and its adjacent sequences in widely divergent species.

Eco RI restriction endonuclease DNA fragments from several representatives of the kingdoms Protista and Animalia were electrophoretically separated and transferred to the nitrocellulose filters. These DNA's were hybridized with [32p]-labelled actin coding sequence from Drosophila melanogaster (Dm). The results indicate that the nucleic acid sequences of the genes coding for actin(s) has been highly conserved throughout evolution. Similar experiments were performed using the sequence derived from the 5' end of Drosophila actin gene as a probe. Cross-hybridization was observed between Drosophila and Acanthamoeba castellanii. This may indicate a functionally important region at the 5' end which has been conserved.

Comparison of circulating colony-forming cells in chronic granulocytic leukemia and leukemoid reaction.

In vitro culture studies of peripheral blood leukocytes using semi-solid media from 8 patients with chronic granulocytic leukemia (CGL) and 5 patients with granulocytic leukemoid reaction were performed. A markedly increased number of circulating colony-forming units were present in patients with CGL (mean 343 +/- 47) as opposed to those having granulocytic leukemoid reaction (mean 7.0 +/- 4). The colony size was larger in CGL than in granulocytic leukemoid reaction or in normal peripheral blood.

Overlapping deficiencies refine the map position of the sex-linked actin gene of Drosophila melanogaster.

A 3H-labelled actin-specific probe was hybridized to Drosophila melanogaster X chromosomes heterozygous for deficiencies in the 5C region. The results suggest that the sex-linked actin gene resides in the overlap region of Df (1) C149 and Df (1) N73 at 5C3-4.