Phylogenetic relationships of Iranian Allium sect. Allium (Amaryllidaceae,Allioideae) as inferred from nrDNA ITS,cpDNA rps16 and trnL–F sequences

Allium is a particularly species rich (more than 800 species) and economically important genus, with numerous taxonomic problems at all levels of classification. In this study, we try to uncover the phylogenetic relationships of the common leek Allium ampeloprasum based on selected samples of this species and its putative relatives in A. sect. Allium from Iran. The silica‐dried leaf samples of 56 accessions representing 23 species of Allium were sequenced; 53 sequences of nrDNA ITS, 35 sequences of plastid rps16 and 52 sequences of trnL–F were generated and additional accessions were extracted from GenBank in order to cover all recognized main lineages in the genus. Maximum Parsimony and Bayesian Inference generated similar trees, but the placement of A. ampeloprasum and its relatives differed slightly between the nuclear and the plastid phylogenies. In the nrITS tree, A. ampeloprasum is retrieved as a highly supported clade with A. iranicum, while in the combined plastid tree A. ampeloprasum formed a highly supported clade with A. vineale. This supports the hypothesis of a possible hybrid origin of A. ampeloprasum. Allium iranicum formed a clade in the plastid tree, but was resolved as paraphyletic in the nrITS tree, probably due to presence of multiple non‐concerted copies of nrITS. Close relationships are suggested between the following species: A. aznavense and A. wendelboi with A. talyschense, A. erubescens and A. rotundum with A. scorodoprasum and A. abbasii with A. phanerantherum.     

The effects of infant births on the sociosexual behavior and hormonal patterns of a cooperatively breeding primate (Cebuella pygmaea)

This study examined changes in the behavioral and hormonal patterns of cooperatively breeding pairs in a primate species with the passing of time and with specific reproductive events. We (1) compared patterns of sexual, agonistic, and affiliative behavior of newly paired pygmy marmosets with the same behavioral patterns immediately after the birth of their first set of infants; (2) determined if postpartum behavioral differences existed between pairs whose infants lived and those whose infants died; and (3) examined whether behavioral patterns changed over the course of ovarian cycles in parous pygmy marmosets as had been documented in nulliparous pairs. The behavior of pairs was recorded during daily half-hour focal samples for 60 days after pairing, and 30 or 60 days after the birth of infants for pairs whose litters died or lived, respectively. Daily urine samples from females during the study were analyzed for luteinizing hormone and pregnanediol glucuronide concentrations to determine dates of ovulation. The results indicated that males consistently altered their sexual behavior and olfactory monitoring of mates during periovulatory periods in the females' cycles both postpairing and postpartum, while similar rates of social and sexual behavior were maintained between the conditions. Sexual behavior occurred throughout the females' ovarian cycles. Peaks in sexual behavior during the periovulatory period in nulliparous pairs disappeared after the birth of infants. Pairs whose infants died showed higher rates of sexual behavior than pairs with surviving infants. Social and sexual behavior may function to maintain the relationship both during and outside of ovulation, especially with the loss of infants. © 1996 Wiley-Liss, Inc.     

Experimental Models of Protein–RNA Interaction: Isolation and Analyses of tRNAPhe and U1 snRNA-Binding Peptides from Bacteriophage Display Libraries

Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNA^Phe (tRNA _AC ^Phe ) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (K _d ? 0.1–5.0 μM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein–RNA complexes.     

Genomic rearrangements at the <Emphasis Type="Italic">IGHMBP2</Emphasis> gene locus in two patients with SMARD1

Autosomal recessive spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by mutations in the immunoglobulin -binding protein 2 (IGHMBP2) gene. Patients affected by the infantile form of SMARD1 present with early onset respiratory distress. So far, patients with neither juvenile onset nor with larger deletions/rearrangements in IGHMBP2 have been reported. In this study, we investigated one patient with infantile (4 months) and another with juvenile (4.3 years) onset of respiratory distress. Direct sequencing of all exons and flanking intron sequences in both patients revealed a mutation on only one allele. In both patients, we identified genomic rearrangements of the other allele of IGHMBP2 by means of Southern blotting. Putative breakpoints were confirmed by polymerase chain reaction on genomic and cDNA. The patient with juvenile onset had an Alu/Alu mediated rearrangement, which resulted in the loss of ~18.5 kb genomic DNA. At the mRNA level, this caused an in-frame deletion of exons 3–7. The patient with infantile onset had a complex rearrangement with two deletions and an inversion between intron 10 and 14. This rearrangement led to a frameshift at the mRNA level. Our results show that SMARD1 can be caused by genomic rearrangements at the IGHMBP2 gene locus. This may be missed by mere sequence analysis. Additionally, we demonstrate that juvenile onset SMARD1 may also be caused by mutations of IGHMBP2. The complex nature of the genomic rearrangement in the patient with infantile SMARD1 is discussed and a deletion mechanism is proposed.     

Bipolar anastomosis technique with removable instruments: an easy, fast, and reliable technique for vascular anastomosis

The interrupted suture technique is most commonly used for microsurgical vascular anastomosis. For several reasons (e.g., exposure of suture material to blood, time needed), many attempts have been made to find other solutions. This article describes a new means of performing a microsurgical vascular anastomosis. The aim of this study was to show the feasibility and possible advantages of this new technique. The basic components at work here are a modified cuff and electrically generated heat used to unite the vessel walls. In this way, both endothelial layers are adapted without manipulating the inside of the vessel or leaving behind foreign matter. Various energy/coagulation time settings were used to perform arterial anastomoses (n = 42) in an isogeneic abdominal aorta interposition model in the rat. The quality of anastomosis was evaluated at days 1, 10, 21, and 120. Immediately after the welding process all anastomoses (n = 42) were patent. No stenosis was found at any observation time. Anastomosis time ranged from 3 to 18 minutes (average, 11 minutes). This new technique permits a vascular anastomosis to be performed easily and reliably with a high patency rate. With this technique, the authors are convinced that a skilled surgeon can create a high-quality anastomosis in a fraction of the time needed to sew an anastomosis.     

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.     

FFA cause hepatic insulin resistance by inhibiting insulin suppression of glycogenolysis

Free fatty acids (FFA) have been shown to inhibit insulin suppression of endogenous glucose production (EGP). To determine whether this is the result of stimulation by FFA of gluconeogenesis (GNG) or glycogenolysis (GL) or a combination of both, we have determined rates of GNG and GL (with (2)H(2)O) and EGP in 16 healthy nondiabetic volunteers (11 males, 5 females) during euglycemic-hyperinsulinemic (~450 pM) clamping performed either with or without simultaneous intravenous infusion of lipid plus heparin. During insulin infusion, FFA decreased from 571 to 30 micromol/l (P < 0.001), EGP from 15.7 to 2.0 micromol x kg(-1) x min(-1) (P < 0.01), GNG from 8.2 to 3.7 micromol x kg(-1). min(-1) (P < 0.05), and GL from 7.4 to -1.7 micromol x kg(-1). min(-1) (P < 0.02). During insulin plus lipid/heparin infusion, FFA increased from 499 to 1,247 micromol/l (P < 0.001). EGP decreased 64% less than during insulin alone (-5.1 +/- 0.7 vs. -13.7 +/- 3.4 micromol x kg(-1). min(-1)). The decrease in GNG was not significantly different from the decrease of GNG during insulin alone (-2.6 vs. -4.5 micromol x kg(-1). min(-1), not significant). In contrast, GL decreased 66% less than during insulin alone (-3.1 vs. -9.2 micromol x kg(-1). min(-1), P < 0.05). We conclude that insulin suppressed EGP by inhibiting GL more than GNG and that elevated plasma FFA levels attenuated the suppression of EGP by interfering with insulin suppression of GL.     

Laser-based micromanipulation for separation and identification of individual Frankia vesicles

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.     

Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.