Rasheeda S. Zafar

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.

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.

Actin gene expression during embryogenesis of Drosophila melanogaster

Abstract The pattern of actin synthesis during Drosophila melanogaster embryogenesis was examined by following the appearance of actin specific transcripts. Total or poly(A)+RNA from six embryonic stages was separated on methylmercury gels, transferred to diazobenzyloxymethyl (DBM) paper, and hybridized with actin coding and adjacent sequence probes. The results indicate that a 2-kilobase (kb) transcript is present throughout all embryonic stages; however, a second transcript of 1.77 kb is only detected at an appreciable level by 12 hr of development. These transcripts vary relative to each other in the amount and timing of synthesis, thereby indicating that they are independently regulated.

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 proteins 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.

Effect of thyroid hormone (T3)-Responsive changes in surfactant apoproteins on surfactant function during sepsis. Commentary

BACKGROUND Long surfactant phospholipids are altered during sepsis; the role of surfactant apoproteins is unknown. This study investigates the effect of cecal ligation and puncture (CLP) on surfactant functional effectiveness and apoprotein transcriptional activity with or without T3 replacement. METHODS Male Sprague Dawley rats underwent sham laparotomy or CLP with or without T3 replacement. Lung compliance, surfactant adsorption, and surface tension were measured with a surfactometer. Surfactant apoproteins A, B, and C (SP-A, SP-B, SP-C) mRNA was quantified by Northern blot analysis. RESULTS Lung compliance was significantly decreased by sepsis; initial surface tension and adsorption values in CLP animals reflected apoprotein dysfunction. Sepsis decreased SP-A mRNA levels and increased SP-B mRNA; SP-C mRNA were unchanged. T3 treatment improved compliance, adsorption, and ST isotherms in septic animals. CONCLUSION T3 attenuated sepsis-induced surfactant dysfunction and SP-A and SP-B transcriptional changes during sepsis. This suggests an interaction between the thyroid, surfactant apoproteins, and lung surfactant functional effectiveness and requires further study.

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.

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.

Identification of the cDNA for Some of the Polymeric Globins of Glycera dibranchiata

The polymeric fraction of the intracellular Hb of the marine polychaete Glycera dibranchiata consists of at least six components which are different in their amino acid sequences (1,2). Among these sequence differences, two major heterogeneous sequences AMEEKVP and AMNSKVP have been found in residues 117 to 123. To distinguish these two kinds of transcripts, two 17-base oligonucleotide probes (one 32-fold degenerate (MEEKVP) and the other 96-fold degenerate (AMNSKV)) were used to screen the cDNA library. Since the cDNA library was constructed from poly(A+) mRNA of Glycera erythrocytes in pBR322, this screening provided the necessary information to determine if the heterogeneity exists at the mRNA level, and, if it does, what would be its distribution ratio.

The cDNA Sequences Encoding the Polymeric Globins of Glycera dibranchiata

The nucleated erythrocytes of the marine polychaete Glycera dibranchiata, the bloodworm, contain several globin chains of ca. 17 kDa, separable into two Hb fractions (1-3): a “monomeric” fraction and a “polymeric” fraction consisting of self-associated globin chains. Both fractions are present in individual erythrocytes (4). The amino acid sequences of two “monomeric” Glycera globins have been determined, M-II (5) and M-IV (6), and the crystal structure of M-II (7) has been refined recendy to a 1.5 A resolution (8). In the “monomeric” globins, the distal His (E7) is replaced by a Leu residue. Furthermore, the two fractions exhibit extensive heterogeneity with at least five or six distinct components in the “monomeric” fraction (9-15) and a similar number of components in the “polymeric” fraction (16,17).

Book Review / Announcements

Sydney E. Salmona Clinics in Haematology, vol. 11, No. 1 Myeloma and Related Disorders Saunders, Eastbourne 1982 VII + 238 pp.; E 10.75, bound ISSN 0308-2261 Within the 238 pages of this very legible text the authors have presented a broad, yet concise review of the current concepts of myeloma in the clinic and in the laboratory. The clinician concerned with the treatment of myeloma patients is provided with guidelines in distinguishing between disease categories requiring systemic treatment, the localized and extramedullary plasmacytomas best treated locally and those with monoclonal gammopathy of undetermined significance (MGUS) probably best not treated at all initially. The prognosis and therapeutic results to be expected for these various types of presentation are taken from the authors own experience and cooperative group studies. Insight into the future prospects of treatment are given not only for currently available cytostatic agents, but also for interferon as well as in vitro cloning and chemosensitivity. Such prospects are also hinted at in the chapter on immunoregulato-ry circuits in myeloma. Those seeking fundamental information regarding the pathogenic mechanisms involved in the frequent complications of this disease such as infection and humoral immunoinsufficiency, hypercalcemia, and renal insufficiency will be rewarded. The chapter on amyloidosis depicts this disease in its various forms as to clinical presentation, diagnosis, laboratory findings, and therapeutic possibilities. Last but not least, the chapter on plasma cell neoplasms and acute leukemia places the connection of these two entities in its proper perspective according to present-day knowledge and experience. The possible role of cytostatic treatment in this relationship, either direct or indirect, is discussed in detail. This book provides the student of this disease in any or all of its aspects with enjoyable and informa tive reading. The ample list of references with each chapter should more than satisfy all readers requir ing more detail. R. Sonntag, Bern Buenos Aires, Argentina, September 1-7, 1984 President: Prof. L.J. Bergna