Limb regeneration of the adult newt (Notophthalmus viridescens) in the absence of the spleen

Summary It has been suggested that the immune system might figure prominently in the regulation of forelimb regeneration. However, neither the nature of this influence nor the aspect(s) of regeneration influenced are clearly known. The determination of which components of the immune system are indispensable for regeneration would be a logical first step in attempting to address such questions. This investigation, therefore, examined the effects of removing the spleen, a major lymphoid organ in the newt, upon the progress of regeneration. Splenectomies performed concomitantly with or after forelimb amputation failed to alter the time course of regeneration. Splenectomies, but not sham-splenectomies, performed prior to amputation reduced the time required to achieve successive stages of regeneration under some, but not all conditions, i.e., when performed 10–20 days before amputation, during the late fall and winter. Up until 35 days after amputation, no gross morphological distortions were observed as a result of splenectomy. It was concluded that the spleen is not required for regeneration to occur.Portions of this work constitute part of the thesis submitted by M.E. Fini in partial fulfillment of the requirements for the M.S. degree in Biology at Boston College     

Concanavalin A induced inhibition of 5'-nucleotidase from guinea pig skeletal muscle and bull seminal plasma: a comparative study

Both purified and membrane-bound 5'-nucleotidases (EC 3.1,3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (K'i = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K'i = 50 nM). 5'-Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i = Ki = 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K'i greater than Ki; 30 and 14 nM, respectively). The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of alpha-methyl-D-mannoside.     

CD38 molecule: structural and biochemical analysis on human T lymphocytes, thymocytes, and plasma cells.

The structure of the CD38 molecule has been evaluated by one- and two-dimensional gel analysis and by enzymatic digestions. The source of the Ag was mainly membrane preparations obtained from MLC cells, from normal thymocytes, and from the plasmocytoma line LP-1. Membranes were solubilized in NP-40 and the extracts fractionated by immunoaffinity chromatography [using a specific anti-CD38 antibody (A10 mAb) covalently linked to Sepharose protein A]. The purified Ag migrated as a single chain of Mr = 45,000 not associated with beta 2-microglobulin. Two-dimensional IEF gel electrophoresis revealed five spots (isoelectric point (pI) range: 6.5 to 6.9). After neuraminidase treatment, the mobility of the five polypeptides shifted to a more basic pI. Endoglycosidase-H treatment reduced the Mr of CD38 by 20%, revealing a broader band centered at Mr = 36,000. Treatment of CD38 molecule with V8 Staphylococcus aureus protease yielded a single dominant band at Mr = 38,000 which was still reactive with A10 mAb. The CD38 molecular was trypsin-resistant in both denatured or native conditions. These results clearly show the glycoprotein nature of CD38 molecule, which includes 2 to 4 N-linked oligosaccharide chains containing sialic acid residues. Furthermore, the present data indicate that the CD38 molecule does not display an apparent biochemical polymorphism among the different CD38+ cells or lines.     

NMR structural characterization of the reaction product between d(GpG) and the octahedral antitumor complex trans-RuCl2(DMSO)4.

The reaction between the antitumor octahedral complex trans-RuCl2(DMSO)4 and d(GpG) leads to the formation of a stable compound characterized by a covalent bifunctional coordination of the bases to the metal center. The structure of the compound has been fully characterized by NMR and molecular modeling studies, showing the presence of two N7-coordinated guanine moieties in a head to head conformation, two dimethyl sulfoxide molecules, and one halogen atom in the coordination sphere of the ruthenium. The glycosidic chi angles are essentially in the anti range, the sugar puckering of the 5'G is 3'-endo (100% N), whereas that of the 3'G is more flexible but mainly in 2'-endo conformation (85% S), the two bases are strongly destacked. The compound shows structural features which are surprisingly similar to those exhibited by the corresponding cisplatin complex, indicating that such a way of interaction with DNA is not exclusive to Pt or to metals with square planar coordination geometries.     

Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

Glycoalkaloid Profile in Potato Haploids Derived from Solanum tuberosum–S. bulbocastanum Somatic Hybrids

Cultivated and wild potato species synthesize a wide variety of steroidal glycoalkaloids (GA) that may affect either human health or biotic stress resistance. Therefore, GA composition must be a major criterion in the evaluation of breeding products when species genomes are merged and/or manipulated. This work reports the results of GA analysis performed on unique haploid (2n=2x=24) plants obtained from tetraploid (2n=4x=48) Solanum bulbocastanum–S. tuberosum hybrids through in vitro anther culture. Glycoalkaloids were extracted from tubers and analyzed by HPLC. Haploids generally showed the occurrence of parental GA. However, in several cases loss of parental GA and gain of new GA lacking in the parents was observed. It may be hypothesized that new GA profiles of our haploids is the result of either genetic recombination or combinatorial biochemistry events. To highlight differences between haploids and parents, soluble proteins and antioxidant activities were also determined. Both were always higher in haploids compared to their parents. The nature of the newly formed GAs will be further investigated, because they may represent new metabolites that can be used against pest and diseases, or are useful for human health.     

An extracellular vesicle epitope profile is associated with acute myocardial infarction

The current standard biomarker for myocardial infarction (MI) is high‐sensitive troponin. Although powerful in clinical setting, search for new markers is warranted as early diagnosis of MI is associated with improved outcomes. Extracellular vesicles (EVs) attracted considerable interest as new blood biomarkers. A training cohort used for diagnostic modelling included 30 patients with STEMI, 38 with stable angina (SA) and 30 matched‐controls. Extracellular vesicle concentration was assessed by nanoparticle tracking analysis. Extracellular vesicle surface‐epitopes were measured by flow cytometry. Diagnostic models were developed using machine learning algorithms and validated on an independent cohort of 80 patients. Serum EV concentration from STEMI patients was increased as compared to controls and SA. EV levels of CD62P, CD42a, CD41b, CD31 and CD40 increased in STEMI, and to a lesser extent in SA patients. An aggregate marker including EV concentration and CD62P/CD42a levels achieved non‐inferiority to troponin, discriminating STEMI from controls (AUC = 0.969). A random forest model based on EV biomarkers discriminated the two groups with 100% accuracy. EV markers and RF model confirmed high diagnostic performance at validation. In conclusion, patients with acute MI or SA exhibit characteristic EV biomarker profiles. EV biomarkers hold great potential as early markers for the management of patients with MI.     

Plant respiration: Controlled by photosynthesis or biomass?

Two simplifying hypotheses have been proposed for whole‐plant respiration. One links respiration to photosynthesis; the other to biomass. Using a first‐principles carbon balance model with a prescribed live woody biomass turnover, applied at a forest research site where multidecadal measurements are available for comparison, we show that if turnover is fast the accumulation of respiring biomass is low and respiration depends primarily on photosynthesis; while if turnover is slow the accumulation of respiring biomass is high and respiration depends primarily on biomass. But the first scenario is inconsistent with evidence for substantial carry‐over of fixed carbon between years, while the second implies far too great an increase in respiration during stand development—leading to depleted carbohydrate reserves and an unrealistically high mortality risk. These two mutually incompatible hypotheses are thus both incorrect. Respiration is not linearly related either to photosynthesis or to biomass, but it is more strongly controlled by recent photosynthates (and reserve availability) than by total biomass.