Glycine hydrazide as a bifunctional reagent for the synthesis of antigens with steroids as the immunodeterminant groups (author's transl)]

The suitability of glycine hydrazide as a link between steroids and carrier proteins in the synthesis of antigens was examined. Testosterone was used as hapten; bovine serum albumin as carrier protein. The reaction described here of testosterone with glycine hydrazide to form testosterone glycylhydrazone acetate took place under milk conditions and the yield was nearly quantitative. Rabbits immunized with the new antigen developed specific antibodies against testosterone.     

Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

Prokaryotic toxin-antitoxin stress response loci

Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.     

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.     

Differences in regional capillary distribution and myocyte sizes in normal and hypertrophic rat hearts.

Data are reported which show significant regional capillary differences in left ventricular endocardium and epicardium of normal rats and of rats with hyperthyroid-induced cardiac hypertrophy. The epicardial region of control rats has 38% more capillaries than the endocardial region. Control endocardial myocytes are 62% larger in cross-sectional area than epicardial myocytes. Hypertrophic hearts exhibit regional differences in capillary density similar to those in the normal hearts, but there is an overall reduction of 12 and 17.5% in capillary density in both regions. The average cross-sectional area of myocytes increases 34.5% in the epicardium and 22.5% in the endocardium.     

Response of Antarctic benthic communities to disturbance: first results from the artificial Benthic Disturbance Experiment on the eastern Weddell Sea Shelf, Antarctica

The long-term benthic disturbance experiment (BENDEX) was started on the eastern Weddell Sea shelf off Austasen (Antarctica) during ‘Polarstern’ cruise ANT XXI/2 in December 2003 to simulate the impact of grounding icebergs on the seabed and follow the steps and timescales of recovery of disturbed benthos and demersal fish communities. Here, we report the basic approach and first results for this experimental field study. By means of 11 densely-placed hauls with a modified bottom trawl, a seabed area of approximately 100 × 1000 m was artificially scoured to inflict a similar damage to the benthic habitats as a grounding iceberg. Before the disturbance event and 11 days after it, the seafloor communities were sampled (invertebrate assemblages by multibox corers, the fish fauna by trawl hauls) and comparatively analyzed. Sediment texture and chemistry was not significantly altered by the heavy disturbance inflicted by repeated trawling, whereas the fauna was negatively affected. Invertebrate benthic biomass was drastically reduced by a factor of 10, while mean abundances were only slightly reduced. Demersal fish biomass and abundance were slightly but not significantly smaller after the disturbance. Effects of disturbance became more evident in the composition of the fish fauna, with Trematomus pennelli and T. hansoni being dominant at disturbed sites, whereas Chionodraco myersi was the dominant species in trawl catches from undisturbed stations.     

Interaction of phenolsulphonphthalein dyes with rabbit plasma and rabbit serum albumin.

Interaction of various substituted phenolsulphonphthalein dyes to rabbit plasma and rabbit serum albumin has been studied by ultrafiltration, equilibrium dialysis and spectrophotometry. The results obtained by ultrafiltration and equilibrium dialysis showed that the degree of binding of these dyes to protein increases in the following order: Phenol red less than bromophenol blue less than bromocresol green less than bromothymol blue. Analysis of binding results revealed that five molecules of bromothymol blue bound very strongly to a molecule of rabbit albumin, whereas only two and three molecules of bromophenol blue and bromocresol green strongly interact with the protein, respectively. It is suggested that strong binding of these substances to protein may be related to the hydrophobicity of these compounds. Finally, an attempt has been made to evaluate the possibility, whether the spectral changes occurred during interaction of dyes to albumin can be utilized for the determination of binding of these ligands to proteins.