Proteinase inhibitory activity in the plasma of a mollusc: evidence for the presence of alpha-macroglobulin in Biomphalaria glabrata.

1. A methylamine-sensitive inhibitor was present in the plasma of B. glabrata. 2. This inhibitor decreased trypsin activity against a protein substrate, however trypsin retained activity against a low molecular weight substrate in the presence of the inhibitor. 3. Snail plasma protected trypsin from inhibition by soybean trypsin inhibitor. 4. The results give evidence for an alpha-macroglobulin proteinase inhibitor in the plasma of this gastropod mollusc.     

Fish cell lines: Establishment and characterization of nine cell lines from salmonids

Summary Nine permanent cell lines have been established from five species of salmonids native to America's Pacific Northwest. With the exception of a hepatoma from an adult trout, the lines were derived from normal tissues of embryonic or juvenile fish. Cells were routinely grown in Eagle's minimum essential medium with 10% fetal bovine serum. Optimum growth temperatures for these lines ranged from 21 to 24°C. All survived storage for at least 1 yr at −65°C and at least 5 yr in liquid nitrogen. Six of the lines were demonstrably free of any microbial contamination but mycoplasmas were found in three. Eight of the lines were heteroploid. The morphology of only one was fibroblastic. All the lines effectively replicated one or more of the common salmonid viruses. Isozyme patterns were consistent with those of the species of origin. These cell lines have significant application in fish virology. This work is a result of research sponsored in part by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant NA79AA-D-0016 and by the Oregon Department of Fish and Wildlife under PL-89304 Anadromous Fish Act and is Oregon Agricultural Experiment Station Technical Paper 6857.     

Reflections on arthropod evolution

Recent claims that arthropods are monophyletic because all have jaws composed of a five-segmented coxa, that the groundplan of arthropod legs has no less than 11 segments, that crustaceans, chelicerates and insects share a 'polyramous arthropod leg', and that the labrum is formed from a pair of legs, are rejected on factual grounds. It is suggested that the earliest arthropod appendages were unsegmented. Putative homologies among mandibulate arthropods are considered. Striking as some of these are, a good case can be made for their convergent evolution, and the concept of the Mandibulata is rejected. Suggested separate ancestries of crustaceans and tracheates are compared. A realistic explanation of radiation from a common arthropod ancestor remains illusory. A polyphyletic concept of arthropod evolution from soft-bodied, segmented, haemocoele-possessing, non-annelid worms is elaborated. The degree of convergence demanded is amply matched by proven examples of the phenomenon. If the earliest arthropods lacked compound eyes, and these were acquired several times, as they have been at least twice in non-arthropods, several otherwise intractable problems are resolved. Sequence comparisons provide a powerful tool for determining relationships but seem powerless to establish whether arthropods are monophyletic, or polyphyletic in the manner envisaged here.     

An endogenous factor induces heterogeneity of binding sites of selective muscarinic receptor antagonists in rat heart

According to molecular biological and pharmacological criteria, rat heart membranes normally express only one muscarinic receptor subtype. The selective antagonists pirenzepine and AF-DX 116 bind to this receptor with a single affinity: low and high, respectively. We report here that an endogenous, intracellular factor alters the affinity of selective antagonists for muscarinic receptors in the heart. Thus, when the intracellular fluid is added back to rat heart membranes, both pirenzepine and AF-DX 116 bind to two receptor sites. Approximately 30% of the receptors bind pirenzepine with high affinity and AF-DX 116 with low affinity. Thus, while cardiac muscarinic receptors are coded for by a single mRNA and are therefore genetically homogeneous, the resulting receptor protein might behave like a mixture of receptor subtypes in intact tissues due to the influence of intracellular factors on receptor conformation.     

The high molecular weight Cat-301 chondroitin sulfate proteoglycan from brain is related to the large aggregating proteoglycan from cartilage, aggrecan.

Monoclonal antibodies Cat-301 and Cat-304 recognize a neuronal cell surface-associated chondroitin sulfate proteoglycan (CSPG), which is expressed during critical periods of postnatal development in the mammalian central nervous system (CNS). In the present study we show that the CNS CSPG identified by Cat-301/304 is similar to aggrecan, the high molecular weight CSPG from cartilage. By Western blot analysis, cartilaginous tissues, which are rich sources of aggrecan, have a high concentration of a high molecular weight CSPG which is immunoreactive with Cat-301 and 304. The Cat-301 and 304 epitopes, however, are partially masked by chondroitin sulfate glycosamino-glycan and are unmasked by digestion of the antigen with chondroitinase ABC. Although the antigen from both cartilage and CNS can be purified by CsCl buoyant density gradient centrifugation, a standard technique for purifying aggrecan, most of the antigen from the CNS has a lower buoyant density than that of cartilage. This may be due, in part, to the paucity of keratan sulfate substitution on the CNS antigen compared with that of the cartilage antigen. Both the CNS and cartilage antigens bind to hyaluronic acid, a feature characteristic of aggrecan. The physiochemical, biochemical, and functional properties of the Cat-301/304 antigen from cartilage are identical to aggrecan. The CNS antigen is similar, but not identical, to the cartilage antigen, and may thus represent another member of the family of high molecular weight CSPGs which bind to and aggregate with hyaluronic acid.     

The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.     

Biological implications of a suggested Late Pleistocene desiccation of Lake Victoria

If, as recently suggested, Lake Victoria dried up completely in theLate Pleistocene and refilled c 12,400 BP, not only must therate of speciation of its flock of cichlid fishes have been extremelyrapid but, more significantly, so too must the rate of morphologicaldifferentiation. Such desiccation also implies that fishes belongingto seven other families achieved endemic status since the lakerefilled, and in one case became generically distinct, acquiredstriking morphological/physiological adaptations to life in deepwater, and split into two species. Such rapid evolution within thesefamilies appears to have no parallel in Africa. This suggests thatprudence be applied in the interpretation of what appears to beunambiguous evidence, especially as this seems to be at variance withwhat also appears to be convincing geophysical evidence of adifferent kind.