The thrombospondin-4 gene

Thrombospondins are a family of extracellular, adhesive proteins that are widely expressed in vertebrates. Five distinct gene products, designated thrombospondin-1 through -4 and cartilage oligomeric matrix protein (COMP), have been identified. With the exception of thrombospondin-4, the structure and location of thrombospondin genes have been determined in the human and/or mouse genomes. In this study, the structure and location of the murine thrombospondin-4 gene and the location of the human thrombospondin-4 gene are reported. The murine thrombospondin-4 gene is approximately 4.5 kb in length and includes 22 exons. Interspecific backcross analysis of progeny derived from matings of (C57BL/6J ×Mus spretus)F₁× C57BL/6J mice indicates that the thrombospondin-4 gene is tightly linked to the Dhfr locus on murine Chromosome (Chr) 13. The human gene maps to Chr 5 in band q13 by in situ hybridization to human metaphase chromosomes. The thrombospondin-4 promoter is similar to promoters of some housekeeping, growth control, and other thrombospondin genes in that it contains multiple GC box sequences and lacks a CAAT box. The presence of multiple E-box sequence motifs is consistent with thrombospondin-4 expression in muscle and bone tissue. Received: 18 May 1998 / Accepted: 24 May 1999     

ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton

rib and raw mutations prevent cells in a number of tissues from assuming specialized shapes, resulting in abnormal tubular epithelia and failure of morphogenetic movements such as dorsal closure. Mutations of zip, which encodes the nonmuscle myosin heavy chain, suppress the phenotypes of rib and raw, suggesting that rib and raw are not directly required for myosin function. Abnormal formation of the actin cytoskeletal structures underlying embryonic cuticular hairs suggests possible roles for rib and raw in organizing the actin cytoskeleton. The actin prehair structures are absent in rib mutants and abnormally shaped in raw mutants, indicating that the two genes have different functions required for organizing the actin cytoskeleton. Received: 4 December 1998 / Accepted: 26 January 1999     

Study of the non-photochemical dark rise in chlorophyll fluorescence in pre-illuminated leaves of various C3 and C4 plants submitted to partial anaerobiosis

Changes in chlorophyll fluorescence yield were studied during a dark period in pre-illuminated leaves of various C3 and C4 plants. The oxygen content in the gaseous atmosphere was either normal (21 kPa) or low (1.5 or 0.36 kPa). C3 and C4 plants of the NAD malic enzyme subgroup showed an initial rise in fluorescence at the onset of the dark period with an amplitude depending on the O₂ level in the gas. In C4 plants belonging to the other two subgroups, the slow rise was absent or of very low size. At high [O₂], the fluorescence level decreased in some minutes to the initial F₀ level (determined in dark-adapted leaves). Conversely at low [O₂], the fluorescence yield remained higher than F₀ in all the C4 plants studied, whereas it decreased slowly to the F₀ level in the different C3 plants. At low [O₂], the fluorescence level decreased rapidly to F₀ when introducing for 30 s, a high O₂ level or when giving a 15-s far-red illumination. At the end of these treatments, the fluorescence level re-increased. These results demonstrate the presence at low [O₂] of highly fluorescent ‘closed' photosystem II centres containing Q^-_A in equilibrium with reduced plastoquinone molecules of the chloroplastic pool. Reoxidation of the plastoquinone pool would be dependent on the functioning of an oxidase probably dependent on a chlororespiration process fully active at O₂ levels higher than 2 kPa. The source of reducing equivalents for the plastoquinone pool is discussed.     

A brief history of myelinated nerve fibers: one hundred and fifty years of controversy

The early controversies over myelinated nerve fibers focused on whether nerves are hollow or not, whether the fatty “marrow” (myelin) is inside the nerve fiber or around it, whether myelin is secreted by the axon or formed by another cell, whether nerve fibers are discrete or part of a syncytial network, whether nodes of Ranvier are present in central myelin or only in peripheral myelin. Since Geren's seminal discovery that peripheral myelin is formed by the Schwann cell plasma membrane wrapped around the axon, the focus has shifted. Myelin is clearly a living cell appendage, and the myelin sheath is dependent upon intercellular interactions not only during its formation, but throughout its lifetime and during pathological processes affecting either the axon or the myelin-forming cell. The myelinated fiber is a functional unit, an exquisite symbiosis, whose ability to perform optimally, in some cases whose very survival, depends on the effects the respective cells exert on one another. How are these interactions mediated? Which structures and functions depend on such interaction and which are independent of it? How do cells of the size and shape of myelin-forming cells cope with their metabolic demands and support their most distal components? What are the mechanisms and mutual consequences of demyelination or axonopathy? Relevant studies have burgeoned with the development of molecular biological and genetic engineering methods, and with improvements in microscopy, in vitro culture and specific immunostaining methods. This introductory essay provides an overview of the structural background and continuing controversies relevant to the articles that follow, which represent a sampling of current work and present new information on the molecular structure, function and pathology of myelin and axoglial interactions.

     

Engineering of betabellin 15D: Copper(II)-induced folding of a fibrillar β-sandwich protein

Summary The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue β-sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, wherep=DPro,k=DLys, andh=DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu²⁺. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited β-sheet structure in the presence of Cu²⁺ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu²⁺ but not in its absence. The observed width of the fibrils (7±2 nm) was consistent with the width (6.8nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D β-sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril.     

Body Fat,Resting and Exercise Blood Pressure and the Angiotensinogen M235T Polymorphism: The Heritage Family Study

RANKINEN, T., JACQUES GAGNON, LOUIS PÉRUSSE, TREVA RICE, ARTHUR s. LEON, JAMES s. SKINNER, JACK H. WILMORE, D. C. RAO, AND CLAUDE BOUCHARD. Body fat, resting and exercise blood pressure and the angiotensinogen M235T polymorphism: the Heritage family study. Obes Res. Objective: The association of resting and exercise blood pressure (BP) and fat mass with the angiotensinogen (AGT) M235T polymorphism was investigated in 522 sedentary Caucasian subjects from 99 families. Research Methods and Procedures: Resting BP was measured on two separate days, three times each day, and the mean of six valid measurements was used. Exercise BP was measured during a cycle ergometer test at a constant power output (50 W). Body composition was derived from underwater weighing and the AGT M235T polymorphism was typed with a polymerase chain reaction-based method. Results: Neither resting nor exercise BP was associated with the AGT genotypes. In mothers, the homozygotes for the T allele showed 8. 8 kg and 7. 1 kg greater (p = 0. 017) age-adjusted body fat mass (FM) than the MM homozygotes and heterozygotes, respectively. Sixty-nine percent of all TT homozygotes were found in the highest FM tertile, whereas only 16% of the MM homozygotes fell in the same tertile (p = 0. 008). Moreover, a significant interaction was seen between FM and T-allele carrier status in women with regard to resting diastolic BP (p = 0. 002). Among women with a FM≥24 kg, carriers of the T allele showed a 6. 3 mmHg higher diastolic blood pressure (DBP) than non-carriers whereas no difference was found in women with a FM less than 24 kg. A similar trend toward an interaction term was evident with resting systolic blood pressure (p = 0. 011) and exercise DBP (p = 0. 012). Body fat was not associated with the AGT polymorphism in fathers or in offspring. Discussion: These data suggest that the AGT M235T polymorphism is associated with body fatness in women, and that the relationship between DBP and AGT M235T polymorphism is dependent on FM in middle-aged sedentary normotensive women.     

The kinetic mechanism of EcoRI endonuclease.

Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with K(m) and k(cat) increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23 M. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spaced EcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a single EcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease.DNA interactions, reflecting facilitated diffusion processes, that occur prior to EcoRI sequence recognition and subsequent to DNA cleavage.