Conserved and non-conserved loci of the glucagon gene in old world ruminating ungulates

The homology and diversification of genomic sequence encoding glucagon gene among native Egyptian buffalos, camel and sheep were tested using cattle as model. Oligodeoxynucleotide primers designed from the available GenBank data were used for PCR probing of the glucagon gene encoding sequence at different loci. The DNA oligomer probes were constructed to flank either the whole gene encoding sequence or different intra-gene encoding sequences. The PCR products were visualized using agarose gel electrophoresis. All species showed a same size band of prepro-glucagon when PCR was used to amplify the whole gene encoding sequence. In contrary, amplifications of different intra-gene loci failed to give the same results. The results indicated variable degrees of diversity among old world ruminating ungulates in the glucagon gene encoding sequence. Compared with other ruminants, the variation appears predominantly in camel. Surprisingly, the similarity in size between both amplification products of whole gene encoding sequence and the proposed size of glucagon cDNA definitely excludes the possibility of large intervening introns spanning the genomic sequence of the glucagon gene in these species. This indicates that, in contrast to other tested mammals, the glucagon gene includes an essentially full-length copy of glucagon mRNA. The study revealed a possible new aspect of glucagon gene evolution in order to correlate its corresponding protein function among different ruminant species.     

In vitro organogenesis and alkaloid accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.     

Increased expression of lipocalin-type prostaglandin D<Subscript>2</Subscript>synthase in osteoarthritic cartilage

Introduction  

Prostaglandin D synthase (PGDS) is responsible for the biosynthesis of PGD and J series, which have been shown to exhibit anti-inflammatory and anticatabolic effects. Two isoforms have been identified: hematopoietic- and lipocalin-type PGDS (H-PGDS and L-PGDS, respectively). The aims of this study were to investigate the expressions of H-PGDS and L-PGDS in cartilage from healthy donors and from patients with osteoarthritis (OA) and to characterize their regulation by interleukin-1-beta (IL-1β) in cultured OA chondrocytes.     

Symbiotic effectiveness and response to mannitol-mediated osmotic stress of various chickpea–rhizobia associations

Thirty-six symbiotic associations involving six chickpea cultivars against six rhizobial strains were evaluated for symbiotic performance and responses to osmotic stress applied by mannitol (50 mM) in aerated hydroponic cultures. Analyses in different symbioses were focused on biomass production, nodulation, nitrogen fixation, and their modulation under osmotic stress conditions, as well as expression of nodular antioxidant enzymes. Mesorhizobium ciceri reference (835) and local (CMG6) strains, as well as the local (C₁₁) M. mediterraneum allowed the best symbiotic efficiency for all chickpea cultivars. The osmotic stress induces severe decrease ranging 30–50% in aerial biomass and 50–70% for nitrogen fixation. Nevertheless, plants inoculated with M. ciceri (835) and M. mediterraneum (C₁₁) preserve a relatively high growth (4 g plant⁻¹) with nitrogen-fixing activity (25 μmols h⁻¹ plant⁻¹). The bacterial partner was the most important factor of variance of the analysed parameters in osmotic stress or physiological conditions where it gets to 60–85%. The strains allowing the best competent symbioses were proposed for field assays. Under osmotic stress, nodular peroxidase (POX) and ascorbate peroxidase (APX) activities were significantly enhanced. The increase of POX and APX was inversely correlated with the inhibition of aerial biomass production (p = 0.05) and nitrogen-fixing capacity (p = 0.01), suggesting a protective role of these enzymes in nodules. Superoxide dismutase (SOD) was also activated in stressed nodules. However, the spectacular decrease in catalase (CAT) activity discounts its involvement in osmotic stress response.     

MSDsite: a database search and retrieval system for the analysis and viewing of bound ligands and active sites

The three-dimensional environments of ligand binding sites have been derived from the parsing and loading of the PDB entries into a relational database. For each bound molecule the biological assembly of the quaternary structure has been used to determine all contact residues and a fast interactive search and retrieval system has been developed. Prosite pattern and short sequence search options are available together with a novel graphical query generator for inter-residue contacts. The database and its query interface are accessible from the Internet through a web server located at: http://www.ebi.ac.uk/msd-srv/msdsite.     

Nectarin IV, a potent endoglucanase inhibitor secreted into the nectar of ornamental tobacco plants. Isolation, cloning, and characterization

We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii x Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5' and 3' rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a K(i) of 0.35 nm. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before.     

Choice of recipient vessels in delayed TRAM flap breast reconstruction after radiotherapy

This study compared the use of the internal mammary and thoracodorsal recipient vessels in a uniform group of patients who underwent delayed TRAM flap reconstruction after radiotherapy, focusing on usability rates and outcomes. The authors identified 123 delayed TRAM flap patients who had undergone postmastectomy radiotherapy from a prospective database (1990 to 2001). Recipient vessel unusability rates were calculated on the basis of reports of inspection of a vessel, either by direct intraoperative dissection or by findings from color Doppler examination (internal mammary vessels only). Charts were reviewed for outcomes including flap loss, vascular complications, fat necrosis, and lymphedema; t-test and chi-square analyses were performed to compare outcomes and unusability rates, and multiple regression analysis was performed to determine factors influencing outcome. Of the 123 planned free TRAM flaps, 106 were completed as free flaps and 17 were performed as pedicled flaps because of unusable recipient vessels. Of the free flaps, 45 were anastomosed to the internal mammary vessels, 55 to the thoracodorsal vessels, and six to other vessels. The internal mammary and thoracodorsal groups did not differ significantly in body mass index, abdominal scars, smoking history, time delay between irradiation and TRAM flap reconstruction, or flap ischemia time. Radiation doses to the axilla (thoracodorsal), internal mammary chain, and supraclavicular fossa were similar between the groups. The internal mammary vessels were rejected in 11 (20 percent) of 56 cases, and the thoracodorsal vessels were rejected in 19 (26 percent) of 74 cases (p = 0.42). In cases with unusable internal mammary vessels, 46 percent (n = 5) had inadequate veins, 27 percent (n = 3) had inadequate arteries, and in 27 percent (n = 3) both vessels were inadequate. In the 19 cases with unusable thoracodorsal vessels, 84 percent (n = 16) were excessively scarred, 11 percent (n = 2) had inadequate vessels, and 5 percent (n = 1) were absent. Outcomes were similar regardless of recipient vessels used (internal mammary versus thoracodorsal): total flap loss, 0 percent versus 4 percent (p = 0.20); vascular complications, 6.7 percent versus 11 percent (p = 0.46); arm lymphedema, 4.4 percent versus 9 percent (p = 0.37); partial flap loss, 9 percent versus 6 percent (p = 0.54); and fat necrosis, 18 percent versus 15 percent (p = 0.69). Multivariate analysis revealed a trend for higher complication rates in smokers and with the use of the thoracodorsal vessels as the recipients. Overall, no discernible unusability or outcome differences were detected between the internal mammary and thoracodorsal groups.     

Inhibition of NOS II prevents cardiac dysfunction in myocardial infarction and congestive heart failure

Strong expression of the inducible form of nitric oxide synthase (NOS II) has been shown in the myocardium of patients with myocardial infarction (MI). We hypothesized that NOS II plays an important role in the development of MI and subsequent heart failure and that inhibition of NOS II may beneficially alter the course of the disease. Long-term administration (2 mo) of the selective NOS II inhibitor S-methylisothiourea (SMT) to rats with MI significantly improved cardiac function. A significant drop in mortality, lung water content, infarct size, and cardiomyocyte hypertrophy was also associated with the use of SMT. Plasma concentration of nitrite and nitrate was also reduced by SMT. Short-term administration of SMT (first 2 wk only) significantly reduced infarct size; however, it did not improve cardiac dysfunction measured 2 mo after MI. These findings demonstrate that induction of NOS II during MI exerts negative effects on cardiac function and structure. Long-term administration of a selective NOS II inhibitor may prove to be beneficial in the treatment of MI and congestive heart failure.     

Autologous breast reconstruction with the extended latissimus dorsi flap

The extended latissimus dorsi myocutaneous flap can provide autogenous tissue replacement of breast volume without an implant. Nevertheless, experience with the extended latissimus dorsi flap for breast reconstruction is relatively limited. In this study, the authors evaluated their experience with the extended latissimus dorsi flap for breast reconstruction to better understand its indications, limitations, complications, and clinical outcomes. All patients who underwent breast reconstruction with extended latissimus dorsi flaps at the authors' institution between January of 1990 and December of 2000 were reviewed. During the study period, 75 extended latissimus dorsi flap breast reconstructions were performed in 67 patients. Bilateral breast reconstructions were performed in eight patients, and 59 patients underwent unilateral breast reconstruction. There were 45 immediate and 30 delayed reconstructions. Mean patient age was 51.5 years. Mean body mass index was 31.8 kg/m2. Flap complications developed in 21 of 75 flaps (28.0 percent), and donor-site complications developed in 29 of 75 donor sites (38.7 percent). Mastectomy skin flap necrosis (17.3 percent) and donor-site seroma (25.3 percent) were found to be the most common complications. There were no flap losses. Patients aged 65 years or older had higher odds of developing flap complications compared with those 45 years or younger (p = 0.03). Patients with size D reconstructed breasts had significantly higher odds of flap complications compared with those with size A or B reconstructed breasts (p = 0.05). Obesity (body mass index greater than or equal to 30 kg/m2) was associated with a 2.15-fold increase in the odds of developing donor-site complications compared with patients with a body mass index less than 30 kg/m2 (p = 0.01). No other studied factors had a significant relationship with flap or donor-site complications. In most patients, the extended latissimus dorsi flap alone, without an implant, can provide good to excellent autologous reconstruction of small to medium sized breasts. In selected patients, larger breasts may be reconstructed with the extended latissimus dorsi flap alone. This flap's main disadvantage is donor-site morbidity with prolonged drainage and risk of seroma. Patients who are obese are at higher risk of developing these donor-site complications. In conclusion, the extended latissimus dorsi flap is a reliable method for total autologous breast reconstruction in most patients and should be considered more often as a primary choice for breast reconstruction.