Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). By virtue of the homopolymeric ribo-tail, the modified cDNA is anchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DNA ligase-dependent ligation. PCR amplification, mediated by two sequence-specific primers, yields the desired unique product suitable for cloning and dideoxy-sequencing.     

Ulstrastructural localization of tumour necrosis factor-alpha

Summary The application of an antibody against tumour necrosis factor-alpha (TNF) to thin sections of plastic-embedded mouse tissue has identified sites of TNF activity in normal and endotoxin-treated C₃N/HeN mice. Prior to endotoxin treatment, TNF was observed in the secretory granules of the antibacterial Paneth cell and one type of crypt endocrine cell. Four hours after endotoxin treatment, these two types of intestinal cell were found to have degranulated. In addition, endotoxin treatment resulted in the appearance of TNF in the secretory granules of all eosinophils, neutrophils and monocytes in the bone marrow, spleen, lung and the proximal intestine. TNF was also observed in the internal elastic lamina (IEL) of arterioles. These results suggest that the process of TNF induction specifically targets the immune system and the vasculature. An invasive stimulus, such as circulating endotoxin, can provoke the immune cells to be armed with TNF. That same stimulus may cause arteriole smooth muscle cells to secrete TNF. TNF secretion in the presence of arteriole smooth muscle cells may play a role in the adjustment of arteriole tone. In the venules, TNF may be responsible for platelet and neutrophil accumulation which leads to embolism formation.     

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B₁ and B₂. The cDNAs encoding functional B₂ receptors have recently been cloned, but no molecular information exists at present on the B₁ receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B₁ and B₂ types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B₁ and B₂ receptors. In this report, we describe oocyte expression experiments showing that the B₁ receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B₂ receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B₂ sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B₁-type pharmacology may arise from two independent molecular mechanisms.     

Genetic Variation and Random Drift in Autotetraploid Populations

The rate of decay of genetic variation is determined for randomly mating autotetraploid populations of finite size, and the equilibrium homozygosity under mutation and random drift is calculated. It is shown that heterozygosity is lost at a slower rate than in diploid populations, and that the equilibrium heterozygosity with mutation and random drift is higher than for diploids. Outcrossing populations as well as populations that randomly self are analyzed. A method of comparing genetic variation between autotetraploid and diploid populations is proposed. Our treatment suggests that the ``gametic homozygosity' provides a unified approach for comparing genotypes within a population as well as comparing genetic variation between populations with different levels of ploidy.     

Genome analysis of intergeneric hybrids of apomictic and sexual Australian Elymus species with wheat,barley and rye: implication for the transfer of apomixis to cereals

Summary Four hybrids were obtained between three Australian Elymus taxa and three cereal grains: wheat, rye, and barley. Mean meiotic metaphase-I configurations were 41.14 I, 0.42 rod II, 0.003 ring II, and 0.01 III for E. scabrus var plurinervis x Triticum aestivum (1 hybrid plant), 22.27 I, 2.63 rod II, 0.06 ring II, and 0.12 III for E. scabrus var scabrus x Secale cereale (4 hybrid plants), and 26.65 I, 0.66 rod II, 0.00 ring II, and 0.01 III for E. scabrus var plurinervis x Hordeum vulgare (13 hybrid plants). The I genome of barley also paired very little in a B_III hybrid of apomictic E. rectisetus x H. vulgare (2 hybrid plants). Megasporogenesis in this B_III hybrid was at least facultatively apomeiotic, with the same sort of nuclear elongation, apomeiotic division, and dyad formation seen previously in E. rectisetus itself. All four hybrid combinations were sterile. While spike morphology in the E. scabrus x T. aestivum and E. scabrus x H. vulgare hybrids were intermediate to their parents, E. scabrus x S. cereale and E. rectisetus x H. vulgare looked like their maternal parents.     

Exploring the legacy of African and Indigenous Caribbean admixture in Puerto Rico

Objectives

From an anthropological genetic perspective, little is known about the ethnogenesis of African descendants in Puerto Rico. Furthermore, historical interactions between Indigenous Caribbean and African descendant peoples that may be reflected in the ancestry of contemporary populations are understudied. Given this dearth of genetic research and the precedence for Afro-Indigenous interactions documented by historical, archeological, and other lines of evidence, we sought to assess the biogeographic origins of African descendant Puerto Ricans and to query the potential for Indigenous ancestry within this community.

Materials and Methods

Saliva samples were collected from 58 self-identified African descendant Puerto Ricans residing in Puerto Rico. We sequenced whole mitochondrial genomes and genotyped Y chromosome haplogroups for each male individual (n = 25). Summary statistics, comparative analyses, and network analysis were used to assess diversity and variation in haplogroup distribution between the sample and comparative populations.

Results

As indicated by mitochondrial haplogroups, 66% had African, 5% had European, and 29% had Indigenous American matrilines. Along the Y chromosome, 52% had African, 28% had Western European, 16% had Eurasian, and, notably, 4% had Indigenous American patrilines. Both mitochondrial and Y chromosome haplogroup frequencies were significantly different from several comparative populations.

Discussion

Biogeographic origins are consistent with historical accounts of African, Indigenous American, and European ancestry. However, this first report of Indigenous American paternal ancestry in Puerto Rico suggests distinctive features within African descendant communities on the island. Future studies expanding sampling and incorporating higher resolution genetic markers are necessary to more fully understand African descendant history in Puerto Rico.     

Respiratory muscle reserve in rats during heavy exercise

Gosselin, Luc E., David Megirian, Joshua Rodman, DonnaMueller, and Gaspar A. Farkas. Respiratory muscle reserve in ratsduring heavy exercise. J. Appl.Physiol. 83(4): 1405-1409, 1997.The extent towhich the respiratory pump muscles limit maximal aerobic capacity inquadrupeds is not entirely clear. To examine the effect of reducedrespiratory muscle reserve on aerobic capacity, whole bodypeak oxygen consumption(O_{2 peak}) wasmeasured in healthy Sprague-Dawley rats before and after Sham,unilateral, or bilateral hemidiaphragm denervation (Dnv) surgery.O_{2 peak} wasdetermined by using a graded treadmill running test.Hemidiaphragm paralysis was verified after testing byrecording the absence of electromyographic activity duringinspiration. Before surgery, O_{2 peak} averaged 86, 87, and 92 ml · kg¹ · min¹for the Sham, unilateral, and bilateral Dnv groups, respectively. Twoweeks after surgery, there was no significant change inO_{2 peak} foreither the Sham or unilateral Dnv group. However,O_{2 peak} decreased~19% in the bilateral Dnv group 2 wk after surgery. These findingsstrongly suggest that the pulmonary system in rats is designed suchthat during heavy exercise, the remaining respiratory pump muscles areable to compensate for the loss of one hemidiaphragm, but not of both.

     

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys¹ with Cys¹³ and Cys⁷ with Cys¹⁹, and the side chain of Asp⁹ forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H₂O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR adopted basis Newton Raphson - AIDS acquired immunodeficiency syndrome - CW continuous wave - DMSO dimethylsulfoxide - DQF-COSY two-dimensional double-quantum-filtered correlation spectroscopy - HIV human immunodeficiency virus - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - ppm parts per million - P.E.-COSY two-dimensional primitive exclusive correlation spectroscopy - REDAC redundant dihedral angle constraint - rf radio frequency - rmsd root-mean-square difference - SIV simian immunodeficiency virus - sw spectral width - _m mixing time - TOCSY two-dimensional total correlation spectroscopy - TSP trimethylsilyl-2,2,3,3-²H₄-propionate - 2D two-dimensional