Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine

The production of antibodies (Abs) in chickens and the extraction of specific Abs from egg yolk (IgY Abs) are increasingly attracting the interest of the scientific community, as demonstrated by the significant growth of the IgY literature. This review offers detailed and comprehensive information about IgY-technology, including: a) possibilities for hen keeping in accordance with the Three Rs principles; b) new insights into the IgY transfer mechanism from blood to yolk as a biological basis for the technology; c) the comparative characteristics of IgY Abs and IgG Abs; d) the high efficacy of the technique, in view of the extraordinary amount of IgY Ab produced by one hen in one year (between 20 g and 40 g IgY in total); e) comparisons between the efficacies of IgY Abs and IgG Abs (rabbit, sheep, mouse) in several immunological assays; f) immunisation protocols, as well as the most commonly used IgY-extraction procedures; g) new possibilities for application in human and veterinary medicine, including strategies for the treatment of Helicobacter pylori infection or fatal intestinal diseases in children, particularly in poor countries, for reducing the use of antibiotics, and, in Asia and South America, for producing Abs against snake, spider and scorpion venoms; and h) the use of IgY Abs in various fields of research, also taking into consideration recent developments in South America (particularly Argentina and Cuba) and in Asia.     

SPECIALIZED MEMBRANE JUNCTIONS BETWEEN NEURONS IN THE VERTEBRATE CEREBELLAR CORTEX

"Gap" junctions, the morphological correlate for low-resistance junctions, are demonstrated between some mossy fiber terminals and granule cell dendrites in some lower vertebrate cerebella (gymnotid and frog). Most of the gap junctions (GJs) seen in the gymnotid-fish cerebellum exhibit an asymmetrical configuration, the electron-opaque cytoplasmic material underlying the junction being more extensive in the dendritic than in the axonal side. In the frog cerebellum, the GJs have a symmetrical distribution of such electron-opaque material. In both species the GJs are encountered at the same synaptic interface as the conventional synaptic zone (CSZ), constituting "mixed synapses" in a morphological sense. The axonal surface covered by CSZs is larger than that covered by GJs. In mammalian cerebellum, GJs are observed only in the molecular layer, between perikarya, dendrites, or perikarya and dendrites of the inhibitory interneurons. These GJs are intermixed with attachment plates and intermediary junctions interpreted as simply adhesive. In the mammalian cerebellum, a new type of junction which resembles the septate junctions (SJs) of invertebrate epithelia is observed between axonal branches forming the tip of the brush of basket fibers around the initial segment of the Purkinje cell axon. It is suggested that such junctions may be modified forms of septate junctions. The physiological implications of the possible existence of high-resistance cross-bridges between basket cell terminals, which may compartmentalize the extracellular space and thus regulate extracellular current flow, must be considered.     

d-Aspartate in Human Brain

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.     

Literaturberichte

Ohne Zusammenfassung     

Adoptive immunotherapy of a mouse colon carcinoma with recombinant interleukin-2 alone or combined with lymphokine-activated killer cells or tumor-immune lymphocytes

Summary We have used a BALB/c colonic adenocarcinoma (C-26) to evaluate the therapeutic potential of recombinant interleukin-2 (rIL-2) at high and low dosages in combination with or without lymphokine-activated killers (LAK) or tumor-specific, immune lymphocytes in either an adjuvant spontaneous or an artificial metastasis system. Most (80%) of the mice that underwent s.c. C-26 tumor excision were shown to die of spontaneous metastasis with lung involvement by 1–4 months after excision. Postsurgical systemic treatment with low-dose rIL-2 (3 × 10⁴ U/day, i.p.) increased the survival rate to 31% as compared to 21% (not significant) in excised controls while administration of high-dose rIL-2 (8 × 10⁴ U/day) led to 53% survival (P <0.01). Both LAK cells and C-26-tumor-immune lymphocytes given during rIL-2 treatment significantly increased the effects of rIL-2 at the low but not at the high-dose, with tumor-immune effectors resulting in the highest percentage (63%) of cures. When mice bearing 3-day artificial lung metastases of C-26 cells were treated with low- or high-dose rIL-2, in combination with or without LAK or tumor-immune lymphocytes, a highly significant reduction or abrogation of the number of lung foci was observed with all treatments, including those involving or tumor-immune lymphocytes alone. Assessment of survival benefit in these mice, however, showed survival prolongation, with 20% cures achieved by low-dose rIL-2 alone and up to 65% cures by LAK in combination with low-dose rIL-2. In this system of artificial metastasis high-dose rIL-2 alone increased the survival time but failed to cure the animals, and the addition of LAK was ineffective whereas that of tumor-immune lymphocytes led to 80% cure. These results suggest that tumorimmune lymphocytes are more effective than LAK when combined with rIL-2 and that caution is necessary in extrapolating findings obtained in artificial metastasis models.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

The role of Lys272 in the pyridoxal 5-phosphate active site of Synechococcus glutamate-1-semialdehyde aminotransferase.

Glutamate-1-semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1-semialdehyde to the C1 position to yield the tetrapyrrole precursor 5-aminolevulinate. Based on spectrophotometric and steady-state data, GSA aminotransferase is a B6-containing enzyme which uses a ping-pong bi-bi mechanism described for other aminotransferases. A putative active-site lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding three site directed mutants were expressed in Escherichia coli. The catalytic competence of the resulting enzymes was determined. The similarity of the absorbance spectra of pyridoxal-P-treated forms of Lys272----Arg, Lys272----Ile, Lys272----Glu with free pyridoxal-P indicates that enzyme-bound pyridoxal-P does not form an internal aldimine in in these three site-directed mutants. Whereas Lys----Ile and Lys----Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine-P and pyridoxal-P forms of Lys----Arg induces slow spectral changes, indicating the catalysis of a half-reaction with GSA, 4,5-dioxovalerate and 4,5-diaminovalerate. 5-Aminolevulinate apparently binds with both coenzyme forms of Lys272----Arg, however significant tautomeric rearrangement is only observed with the pyridoxal-P form. It is suggested that Lys272 is the covalent pyridoxal-P-binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5-aminolevulinate. The second-half reaction (4,5-diaminovalerate in equilibrium with 5-aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5-diaminovalerate relative to the enzyme-bound pyridoxal-P.     

Sawfishes and Other Elasmobranch Assemblages from the Mio-Pliocene of the South Caribbean (Urumaco Sequence,Northwestern Venezuela)

The Urumaco stratigraphic sequence, western Venezuela, preserves a variety of paleoenvironments that include terrestrial, riverine, lacustrine and marine facies. A wide range of fossil vertebrates associated with these facies supports the hypothesis of an estuary in that geographic area connected with a hydrographic system that flowed from western Amazonia up to the Proto-Caribbean Sea during the Miocene. Here the elasmobranch assemblages of the middle Miocene to middle Pliocene section of the Urumaco sequence (Socorro, Urumaco and Codore formations) are described. Based on new findings, we document at least 21 taxa of the Lamniformes, Carcharhiniformes, Myliobatiformes and Rajiformes, and describe a new carcharhiniform species (†Carcharhinus caquetius sp. nov.). Moreover, the Urumaco Formation has a high number of well-preserved fossil Pristis rostra, for which we provide a detailed taxonomic revision, and referral in the context of the global Miocene record of Pristis as well as extant species. Using the habitat preference of the living representatives, we hypothesize that the fossil chondrichthyan assemblages from the Urumaco sequence are evidence for marine shallow waters and estuarine habitats.     

Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.