Prolactin and growth hormone stimulation of lactation in mice requires thyroid hormones.

This experiment tested the hypothesis that thyroid hormones are essential for a milk production response to growth hormone (GH) and prolactin (PRL). Prior to breeding, female transgenic mice expressing the herpes simplex type-I thymidine kinase in the thyroid were treated with ganciclovir to ablate thyroid follicular cells. To provide for normal gestation, thyrocyte-ablated mice were supplied thyroxine (T4) in drinking water (0.2 microgram/ml) until 7 days before parturition. Litter size was adjusted to 9 pups, hormone administration began on Day 2 of lactation, and mice were sacrificed on Day 12. There were 5-6 mice in each of 7 treatments that included nonablated controls, thyrocyte-ablated controls, and thyrocyte-ablated mice treated with T4, GH, PRL, GH + T4, and PRL + T4. Thyroxine was administered in drinking water, and GH and PRL (20 microgram/d) were administered by subcutaneous injection. Compared with thyrocyte-ablated controls, litter weight gain was unaffected when dams were treated with GH, PRL, or T4 alone. However, when dams were treated with GH or PRL in combination with T4, litter weight gain increased 13% compared with thyrocyte-ablated controls and 18% compared with GH or PRL-treated mice. Concentration of T4 in serum of pups averaged 62 ng/ml and did not differ among treatments. Concentration of T4 in serum of dams averaged 76 ng/ml when T4-treated. Thyroxine 5'-deiodinase (5'D), the enzyme that converts T4 to triiodothyronine, was quantitated in liver, kidney, and mammary gland. Quantity of 5'D was lower in liver and kidney of thyrocyte-ablated dams without T4 than in respective tissues of mice treated with T4, and there was no effect of GH or PRL. However, in mammary gland, 5'D was increased by treatment with GH, PRL, or T4. Data show that thyroid hormones are necessary for a galactopoietic response to GH and PRL and demonstrate a unique organ-specific regulation of 5'D by galactopoietic hormones.     

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     

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.