Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

The light-harvesting and protective functions of carotenoids in photosynthetic membranes

Siefermann-Harms, D. 1987. The light-harvesting and protective functions of carotenoids in photosynthetic membranes.     

Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of P 0 mRNA in myelin-forming Schwann cells

Summary A biotinylated P ₀ glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P ₀ mRNA. Interestingly, P ₀ mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

Alterations in tissue Mg++, Na+ and spinal cord edema following impact trauma in rats

Alterations in water content and total tissue Na+ and Mg++ of rat spinal cord tissue were followed over time after a 100 g-cm impact injury to the T-9 spinal cord segment. Rats subjected to laminectomy but not trauma served as controls. In the injured segment there was a progressive increase in water content with increased Na+ and decreased Mg++ at 1 hour and 24 hours after trauma. At seven days, water and Na+ content remained elevated, whereas Mg++ levels had returned to preinjury baseline values. Because of its important role in many metabolic and physiological regulatory processes the early decline in Mg++ concentration after trauma may contribute to the development of secondary tissue damage after spinal cord injury.     

A Monte Carlo investigation of homogeneity tests of the odds ratio under various sample size configurations

Epidemiologic data for case-control studies are often summarized into K 2 x 2 tables. Given a fixed number of cases and controls, the degree of sparseness in the data depends on the number of strata, K. The effect of increasing stratification on size and power of seven tests of homogeneity of the odds ratio is studied using Monte Carlo methods. In all the designs considered here, the numbers of cases and controls per stratum are the same. Considering both size and power in non-sparse-data settings, we recommend the Breslow-Day statistic (1980, Statistical Methods in Cancer Research, 1. The Analysis of Case-Control Studies, p. 142; Lyon: International Agency for Research on Cancer) for general use. In sparse-data settings the T4 statistic of Liang and Self (1985, Biometrika 72, 353-358) performs the best when all tables, regardless of sample size, have odds ratios generated from the same distribution. In sparse-data settings characterized by a large table with an odds ratio of 1 and many small tables with odds ratios greater than 1, the T5 statistic of Liang and Self (1985) performs the best. One of the most important results of this study is the generally low power for all homogeneity tests especially when the data are sparse.     

Effects of sulfhydryl reagents on nitroglycerin-induced relaxation of bovine coronary artery

The mechanism whereby nitroglycerin relaxes vascular smooth muscle remains uncertain. A current hypothesis suggests that nitroglycerin reacts with critical cellular sulfhydryl groups to form an intermediate, which activates guanylate cyclase, resulting in cGMP accumulation and relaxation. This study investigated further the potential involvement of sulfhydryls in nitroglycerin-induced vascular smooth muscle relaxation by evaluating effects of a variety of sulfhydryl alkylating and reducing agents on responses to nitroglycerin and other relaxants in bovine coronary arterial strips submaximally contracted using 30 mM K. Whereas 10(-4) M 5,5'-dithiobis-(2-nitrobenzoic acid), 10(-5) MN-ethylmaleimide, and 10(-4) MN-naphthylmaleimide did not affect nitroglycerin-induced relaxation, 10(-4) MN-ethylmaleimide and 10(-4) M ethacrynic acid significantly inhibited relaxation induced by nitroglycerin. Both ethacrynic acid and N-ethylmaleimide at 10(-4) M also inhibited relaxation induced by sodium nitroprusside. N-ethylmaleimide, but not ethacrynic acid, inhibited relaxation induced by isoproterenol and forskolin. Ethacrynic acid significantly reduced both relaxation and cGMP elevation induced by both 10(-7) M nitroglycerin and 10(-7) M sodium nitroprusside. Ethacrynic acid, but not N-ethylmaleimide, significantly reduced relaxation induced by 8-Br-cGMP. Pretreatment with the sulfhydryl-containing agents N-acetylcysteine, 2-mercaptoethanol, or dithiothreitol, at 10(-3) M did not affect nitroglycerin-induced relaxation in nontolerant arteries. Similarly, N-acetylcysteine and dithiothreitol did not alter the depressed responses to nitroglycerin in arteries in which tolerance to nitroglycerin was induced in vitro. A slight but statistically significant reversal of nitroglycerin-tolerance occurred after treatment of tolerant arteries with 2-mercaptoethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex

Recessive lethal mutations in the T/t-complex of the mouse characteristically show defective genetic complementation, even when they affect very different stages of embryogenesis and are known to be nonallelic. To address the question of their genetic or functional relationship, we have applied the cis-trans test, using several well defined recombinant t-chromosomes that carry two or more lethal mutations, and others that are devoid of specific lethals. We show here that the defective complementation that occurs between different t-lethals is a specific result of the trans configuration; thus these genes, which may map as much as 15 cM apart, constitute a functional unit. Some speculations are presented to interpret this enigma in terms of DNA plasticity.