Ontogeny of the hyobranchial apparatus in the salamanders Ambystoma talpoideum (Ambystomatidae) and Notophthalmus viridescens (Salamandridae): The ecological morphology of two neotenic strategies

Comparison of metamorphosis of skull and hyobranchial system in two species of neotenic salamanders reveals two different types of neoteny. Ambystoma talpoideum is completely neotenic owing to delayed metamorphosis. Notophthalmus viridescens exhibits limited neoteny as a result of incomplete metamorphosis. Morphological details of neoteny are compared to life history in both species in order to discuss the ecological morphology of the two neotenic strategies. Comparisons to Taricha granulosa, Triturus vulgaris, and Ambystoma gracile indicate that these two strategies are widely employed and may represent familial patterns.     

Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.     

Adsorption from fetal calf serum of collagen-like proteins which bind fibronectin and promote cell attachment

Baby hamster kidney cells were seeded onto Western blots of fetal serum proteins which had been extracted from several foreign surfaces. This revealed that the major cell adhesive proteins adsorbed onto these surfaces from fetal serum were (1) fibronectin of Mr 220,000 Da and (2) vitronectin of Mr 65,000 and 78,000 Da. Two minor bands of cell attachment were observed at Mr 153,000 and Mr 134,000 Da in the fetal serum proteins extracted from heparin-agarose and serotonin-agarose. However, by exposing the Western blots of separated proteins to a second round of serum proteins, prior to cell blotting, very strong cell adhesive bands were revealed at Mr 153,000, 134,000, and 120,000 Da. By (i) modifying the composition of the serum proteins used to treat the Western blots, (ii) using specific antibodies to fibronectin, and (iii) using radiolabeled fibronectin, it was conclusively demonstrated that the new cell adhesive bands owed their increased cell attachment activity to secondary binding of fibronectin. The new bands were shown (i) to be trypsin sensitive and collagenase sensitive and therefore to be collagen-like proteins and (ii) to react negatively in immunoblots using anti-fibronectin, anti-vitronectin, anti-fibrinogen, anti-fetuin or anti-thrombospondin. In SDS-PAGE (i) the Mr 120,000-Da protein comigrated with the alpha 2-chain of Type I collagen, (ii) the Mr 134,000-Da protein comigrated with the alpha 1-chain of Type I collagen, and (iii) the Mr 153,000-Da protein comigrated with the pN-alpha 1-chain of Type III collagen. Since the novel collagen-like proteins acted as strong sites of cell attachment on nitrocellulose blots by binding fibronectin, they might well promote cell attachment on the foreign surfaces from which they were extracted.     

Aspergillus myositis in a patient with a myelodysplastic syndrome

We present the case of an elderly man who, while being treated with corticosteroids for a myelodysplastic syndrome, developed myositis of the calf due to Aspergillus fumigatus. Despite therapy with amphotericin B the myositis failed to resolve and he died. At autopsy, a localized necrotizing myositis of the right calf was found with no evidence of disseminated Aspergillus infection. Myositis in the setting of disseminated candidiasis or cryptococcosis has been previously reported. This case is unique in that it is the first reported case of localized fungal myositis and of myositis caused by Aspergillus.     

Heparin inhibition of smooth muscle cell proliferation: a cellular site of action

The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.     

Role of apparent membrane growth initiation sites during photosynthetic membrane development in synchronously dividing Rhodopseudomonas sphaeroides.

Sites of intracytoplasmic membrane growth and temporal relations in the assembly of photosynthetic units were examined in synchronously dividing Rhodopseudomonas sphaeroides cells. After rate-zone sedimentation of cell-free extracts, apparent sites of initiation of intracytoplasmic membrane growth formed an upper pigmented band that sedimented more slowly than the intracytoplasmic membrane-derived chromatophore fraction. Throughout the cell cycle, the levels of the peripheral B800-850 light-harvesting pigment-protein complex relative to those of the core B875 complex in the upper pigmented fraction were only about half those of chromatophores. Pulse-labeling studies with L-[35S]methionine indicated that the rates of assembly of proteins in the upper pigmented fraction were much higher than those of chromatophores throughout the cell cycle; rates for the reaction center polypeptides were estimated to be approximately 3.5-fold higher than in chromatophores when the two membrane fractions were equalized on a protein basis. In pulse-chase studies, radioactivity of the reaction center and B875 polypeptides increased significantly in chromatophores and decreased in the upper pigmented band during cell division. These data suggest that the B875 reaction center cores of the photosynthetic units are inserted preferentially into sites of membrane growth initiation isolated in the upper pigmented band and that the incomplete photosynthetic units are transferred from their sites of assembly into the intracytoplasmic membrane during cell division. These results suggested further that B800-850 is added directly to the intracytoplasmic membrane throughout the cell cycle.     

Purification and characterization of endo-xylanases from Aspergillus niger. II. An enzyme of pl 4.5

A homogeneous endo-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) was obtained from a crude Aspergillus niger pentosanase by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and SP-Sephadex C-25 with a gradient from pH 2.8 to pH 4.6. It was much more active on soluble than on insoluble xylan, yielding large amounts of unreacted xylan and a mixture of oligosaccharides with chain lengths from two to six. No xylose or L-arabinose was produced. There was high activity on a xylopentaose through xylononaose mixture, but not on xylobiose, xylotriose, or xylotetraose. The enzyme had slight activity on untreated cellulose, carboxymethylcellulose, and pectin. Molecular weight was ca. 1.4 x 10(4), with an isoelectric point of 4.5 and an amino acid profile high in acidic but low in sulfur-containing residues. In a 25-min assay at pH 4.7, this endo-xylanase was most active at 45 degrees C, with an activation energy from 5 to 35 degrees C of 33.3 kJ/mol. The optimum pH for activity was 4.9. Decay in buffer was first order, with an activation energy at pH 4.7 from 48 to 53 degrees C of 460 kJ/mol. Optimum pH for stability was about 5.6, where the half-life at 48 degrees C in buffer was ca. 40 h.     

Inactivation of bradykinin and kallidin by cathepsin G and mast cell chymase

Human neutrophil cathepsin G and human skin chymase can inactivate bradykinin by cleavage at the carboxy terminal phenylalanyl-arginyl peptide bond of this polypeptide. The mast cell enzyme is far more effective than cathepsin G, the rates of hydrolysis being comparable to that found for angiotensin I to angiotensin II conversion (C.F. Reilly, D. Tewksbury, N. Schechter, and J. Travis, J. Biological Chemistry 257:8619-8622). This ability to both inactivate bradykinin and accelerate the production of angiotensin II may be of significance in the development of biochemical events associated with inflammation.