Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.     

Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase.

Onconase, a member of the ribonuclease superfamily, is a potent cytotoxic agent that is undergoing phase II/III human clinical trials as an antitumor drug. Native onconase from Rana pipiens and its amphibian homologs have an N-terminal pyroglutamyl residue that is essential for obtaining fully active enzymes with their full potential as cytotoxins. When expressed cytosolically in bacteria, Onconase is isolated with an additional methionyl (Met1) residue and glutaminyl instead of a pyroglutamyl residue at position 1 of the N-terminus and is consequently inactivated. The two reactions necessary for generating the pyroglutamyl residue have been monitored by MALDI-TOF MS. Results show that hydrolysis of Met(-1), catalyzed by Aeromonas aminopeptidase, is optimal at a concentration of >or= 3 m guanidinium-chloride, and at pH 8.0. The intramolecular cyclization of glutaminyl that renders the pyroglutamyl residue is not accelerated by increasing the concentration of denaturing agent or by strong acid or basic conditions. However, temperature clearly accelerates the formation of pyroglutamyl. Taken together, these results have allowed the characterization and optimization of the onconase activation process. This procedure may have more general applicability in optimizing the removal of undesirable N-terminal methionyl residues from recombinant proteins overexpressed in bacteria and providing them with biological and catalytic properties identical to those of the natural enzyme.     

Designing an effective approach for obtaining methylenecarboxylate analogues of adenophostin A. Preliminary results

Preliminary results for the synthesis of two new adenophostin analogues incorporating 3″- and 4″-methylenecarboxylate surrogate groups are presented. The synthesis involves the preparation of 3″- and 4″-methylenecarboxylate glucose derivatives by a radical allylation—oxidative cleavage approach, their conversion into thioglycoside precursors, and stereoselective glycosylation of a suitable adenosine derivative. The glycosylations proceeded with excellent stereoselectivity, only the α product was detected, and in good yields.     

Preparation of hormone-sensitive adipocytes from foetal rat brown adipose tissue

A collagenase-dispersion method involving Percoll gradient separation is described for the production of adipocytes from foetal rat brown adipose tissue. The cells produced are hormone-sensitive as judged by cAMP accumulation and lipolysis.     

Studies on Gonococcal Infection

Macrophages from mice were infected with Neisseria gonorrhoeae type 1 cells, and their ultrastructure was studied by electron microscopy. The macrophages showed various stages of engulfment and digestion of gonococci 2 hr after infection. Infected macrophages seemed to develop pseudopodia for phagocytosis, and could engulf more than 30 gonococcal cells. Some engulfed bacteria appeared morphologically intact, while others appeared lysed and some structures resembling the L form of N. gonorrhoeae were also seen. These observations suggest that gonococcal cells may be able to survive intracellularly with normal or altered forms of morphology, and that macrophages containing these bacteria may disseminate gonococcal infection in man.     

Ultrastructural characterization by ruthenium red of the surface of the fat globule membrane of human and rat milk with data on carbohydrates of fractions of rat milk

The fat globules of the cream fractions of human and rat milk were stained with ruthenium red. Under the electron microscope, discrete granules and an amorphous coat of lesser density are seen at the surface of the milk fat globules. Since ruthenium red binds anionic groups selectively, it is probable that the granules contain the greatest concentration of these groups. The cream fraction of rat milk contains hexoses, hexosamines, methylpentoses and sialic acid. Methylpentoses and hexosamines are significantly enriched in the cream fraction. It is concluded that the finding of a surface coat in milk fat globules is in keeping with the Bargmann-Knoop model and suggests a distinct mechanism for carrying certain complex carbohydrates in milk. The role of the negative charges at the outer surface of the membrane coat is maintaining fat globules in suspension and in binding certain cations such as calcium is suggested.     

Infrared spectra of glycosaminoglycans in deuterium oxide and deuterium chloride solution: Quantitative evaluation of uronic acid and acetamidodeoxyhexose moieties

I.r. spectra in the “carbonyl region” (1800-1400 cm^?1) have been obtained for aqueous solutions of a number of glycosaminoglycans and model compounds. In D₂O solution, the uronic carboxylate and acetamido groups absorb at characteristic frequencies (ν_asCOO^?, 1605 ±5; ν₅COO^?, 1420 ±5; Amide I, 1623 ±3; and Amide II, 1480 ±2 cm^?1). In acidic (m DCl) solution, the amide bands remain substantially unmodified, whereas those of the carboxylate anion disappear and are replaced by a single band due to the undissociated carboxyl (νCOOH, 1723 ±3 cm^?1). These bands can be used for quantitative evaluation of the uronic acid and acetamidodeoxyhexose moieties of glycosaminoglycans, using either standard polysaccharides or the corresponding monosaccharides as reference compounds. For D₂O solutions, the absorbances of the carboxylate and acetamido groups have been measured by graphical extrapolation of the corresponding most-intense bands (ν_asCOO^? and Amide I). In DCl, the two analytical bands (νCOOH and Amide I) are well-resolved, and analyses have been performed directly from the absorbances recorded. I.r. data for the uronic acid and 2-acetamido-2-deoxyhexose content of various reference samples of glycosaminoglycans are in reasonable agreement with those expected from the “established” structures, as well as with those obtained by colorimetric and conductimetric methods. When sodium d-glucuronate was used as the reference standard, the i.r. data gave relatively low values for the uronic acid content of heparin. The apparent acid dissociation constants of chondroitin 4-sulfate and heparin were estimated from the absorbance of νCOO^? and/or the νCOOH bands at different pH (pD) values.     

Blood Vessels and Related Structures in the Gill Bars of Glossobalanus minutus (Enteropneusta)

The branchial circulatory system of Glossobalanus minutus (Enteropneusta) is investigated by means of transmission electron microscopy. Primary gill bars, or septa, have a single blood vessel longitudinally located along the outer edge of the bar. Secondary gill bars, or tongue bars, show a vessel in their inner, pharyngeal edge. The walls of both vessels are made up of the basement membranes of surrounding epithelia, lacking an endothelium. No definite limits between the vessel lumen and the skeletal rods inside the bars can be seen. Furthermore, the blood seems to penetrate into the rods of both primary and secondary gill bars. In the secondary bars such a phenomenon gives rise to the so-called 'lateral vessels' reported in the light microscopical literature. The significance of these observations is discussed, with special reference to the gill circulatory system of amphioxus, which seems to be strongly similar from a morphological and ultrastructural point of view.