The phylogeny of Lens (Leguminosae): new insight from ITS sequence analysis

 Lens includes L. culinaris subsp. culinaris (the cultivated lentil) and several wild species distributed from the Mediterranean region to western Asia. We compared sequence variation in the ITS region among species of Lens in an effort to end persisting uncertainty regarding the phylogeny of the genus. The parsimony analysis revealed a single minimum-length tree with a topology congruent with patterns derived by previous studies of nuclear and chloroplast DNA RFLPs. The basal and highly divergent status of the L. nigricans clade is depicted, and the progenitor-derivative relationship between L. culinaris subsp. orientalis and L. culinaris subsp. culinaris is reaffirmed. Resolution in the tree was improved by combining the ITS data set with a pre-existing set of chloroplast DNA restriction site data obtained from the same group of samples. Received May 8, 2000 Accepted October 26, 2001     

In vitro regeneration of plants from hypocotyl segments of Amaranthus paniculatus

Hypocotyl segments, 5 to 8 mm length from 4 to 7 day old seedlings, callused on B₅ medium supplemented with Kn (0.5 ppm) and NAA (0.1 ppm). Even without transfer, shoots were formed in such cultures. About 20% of the cultures produced multiple shoots. In medium with 1 ppm each of Kn and NAA direct shoots were formed at one end of the hypocotyl segment and callusing was initiated at the other end. The plants obtained in either medium formed roots and could be transferred to soil for further growth.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - Kn Kinetin - NAA naphthalene acetic acid     

Accumulation of 15-Kilodalton Zein in Novel Protein Bodies in Transgenic Tobacco

Zeins, the seed storage proteins of maize, are a group of alcohol-soluble polypeptides of different molecular masses that share a similar amino acid composition but vary in their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal is to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whether this protein would be stable in nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, [beta]-phaseolin, in nonseed tissues and to determine the basis for its stability/instability. We have introduced the 15-kD zein and bean [beta]-phaseolin-coding sequences behind the 35S cauliflower mosaic virus promoter into tobacco (Nicotiana tabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein is stable not only in seeds but in all nonseed tissues tested, whereas the [beta]-phaseolin protein accumulated only in mid- and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the [beta]-phaseolin protein in nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention in the ER or to the protease-resistant nature of the protein.     

Effect of glycaemic control and duration of disease on overnight albumin excretion in diabetic children.

Urine albumin excretion rates were measured in overnight timed samples from diabetic and non-diabetic schoolchildren. The excretion rates in the diabetics were significantly higher than those in the controls and were positively correlated with age, duration of diabetes, and glycaemic control. Diabetic children aged 12 years and older had significantly higher albumin excretion rates than younger diabetic children matched for duration of disease. Among the non-diabetic controls there was no correlation between albumin excretion rate and age and the girls excreted significantly more albumin than the boys. Measurement of the overnight albumin excretion rate may provide a useful early indicator of the progression to clinical proteinuria in diabetes and is free from random variations such as that due to exercise.     

Uterovaginal packing with rolled gauze in postpartum hemorrhage

Management options for postpartum hemorrhage (PPH) include oxytocics, prostaglandins, genital tract exploration, ligation or angiographic embolization of uterine/internal iliac arteries, and hysterectomy. After excluding uterine rupture, genital tract lacerations, and retained placental tissue, efforts are directed toward contracting the uterus by bimanual compression and oxytocics. If these are not successful, one must resort to surgical techniques. At this stage, an alternative option to remember is uterovaginal packing. Easy and quick to perform, it may be used to control bleeding by tamponade effect and stabilize the patient until a surgical procedure is arranged. Uterovaginal packing may sometimes obviate the need for surgery altogether. Two cases, a primary and a secondary PPH, managed recently with uterovaginal packing are reported. Despite concerns about concealed hemorrhage or the development of infection with this intervention, none of these problems were encountered, and uterine packing was successful even in the case of secondary PPH with documented infection.     

The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities

Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.     

Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.