Individual culture and experimental population dynamics of Keratella cochlearis (Rotatoria)

A new culture method for K. cochlearis has made it possible to study isolated animals and to investigate the population dynamics of this pelagic rotifer species. The duration of principal developmental stages diminishes continuously with temperature. Decreased survival was associated with a reduced duration of individual fecundity. The age distribution of the population shifted toward younger age intervals with higher temperatures. Growth rates had an optimum at 15°C; the population dynamics, while lower for K. cochlearis than for some other rotifers, were in good agreement with field data.     

Crystallization of a complex of hemoglobin components II and III of the symbiont-harboring clam Lucina pectinata.

Diffraction data to 3.1 A resolution were collected on crystals of a complex of components II and III of the cytoplasmic hemoglobin of the symbiont-harboring clam Lucina pectinata. The crystal system is tetragonal, a = 76.3 A, c = 153.1 A and the space group is P42212. The asymmetric unit probably contains a dimer of the tetrameric complex.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.     

The complete amino acid sequence of porcine gastrotropin, an ileal protein which stimulates gastric acid and pepsinogen secretion

The stomach is stimulated by an enterooxyntin factor in a delayed response to feeding, resulting in an increase in both gastric acid and pepsinogen secretion. We have previously reported on the identity of such a factor from the porcine ileum (Wider, M. D., Vinik, A. I., and Heldsinger, A. (1984) Endocrinology 115, 1484-1491). This protein, termed gastrotropin, is localized to the distal region of the ileum where it constitutes less than 0.1% of the cytosolic protein. We have completed the primary structure of porcine gastrotropin by Edman degradation and mass spectrometry. Gastrotropin (Mr = 14,054) contains 127 amino acid residues and has a blocked (acetylated) alanine at its NH2 terminus. The sequence of porcine gastrotropin is similar to rat liver fatty acid-binding protein (FABP), with 44 of 127 residues being identical (35%). Homology with other members of the FABP family is significantly less apparent, with the order of similarity being liver FABP greater than heart FABP greater than retinol-binding protein greater than intestine FABP. The sequences of the NH2-terminal regions of these proteins account for virtually all of the homology; there are 9 conserved residues common to all five proteins. Gastrotropin represents the first member of the FABP family which has an extracellular function.     

A new kinin moiety in human plasma kininogens

Recently, we isolated a new kinin from human urine and tentatively identified it as [Ala3]-Lys-bradykinin. However, there were inconsistencies between the properties of the naturally occurring new kinin and synthetic [Ala3]-Lys-bradykinin. In the present work, we determined whether the new kinin was released from human plasma kininogen, and further investigated the structure of the new kinin. After incubation of plasma (n = 6) with human urinary kallikrein, kinins were separated by HPLC and measured by RIA. The new kinin and Lys-bradykinin were found representing 23 +/- 3 and 76 +/- 6%, respectively, of total kinins released (2.0 +/- 0.4 micrograms/ml). The new kinin was also released from both purified low- and high-molecular-weight kininogens, representing 40-42% of total kinins released. Amino acid sequencing and composition analysis indicated that the structure of the new kinin was [Hyp3]-Lys-bradykinin (Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and not [Ala3]-Lys-bradykinin. We conclude that an important proportion of human kininogens contain hydroxyproline instead of proline in position three of the bradykinin moiety.     

Calcium- and inositol polyphosphate-sensitivity of the calcium-sequestering endoplasmic reticulum in the photoreceptor cells of the honeybee drone

Summary The photoreceptor cells in the honeybee drone contain an elaborate Ca²⁺-sequestering endoplasmic reticulum (ER). We measured Ca-oxalate formation within the ER of permeabilized retinal slices with a microphotometer and studied the kinetics of Ca²⁺-uptake into the ER and the properties of Ins(1,4,5)P₃-induced Ca²⁺-release.The ATP-dependent Ca²⁺-uptake mechanism has a high affinity for Ca²⁺: Uptake rate was half maximal at Ca²⁺ _free 0.6 M.Addition of Ins(1,4,5)P₃ caused a persistent depression of Ca-oxalate formation due to Ca²⁺ -release from the ER. The Ins(1,4,5)P₃-dependent Ca²⁺-release mechanism has a high affinity (half maximal rate with 0.2 M Ins(1,4,5)P₃) and a high specificity for Ins(1,4,5)P₃: Ins(2,4,5)P₃ was 6 times, Ins(1,3,4,5)P₄ was 15 times less potent in inducing Ca²⁺-release. 3 M Ins(1,4)P₂ had no detectable effect. The sensitivity for Ins(1,4,5)P₃ was maximal between 280 nM and 1.6 M Ca²⁺ _free and decreased at higher and lower Ca²⁺-concentrations.Our data show that the ER in invertebrate photoreceptor cells is an effective Ca²⁺ -sink and an Ins(1,4,5)P₃-sensitive Ca²⁺-source. We support the idea (Payne et al. 1988) that the ER-network close to the photoreceptive membrane, the submicrovillar cisternae (SMC), are the light- and Ins(1,4,5)P₃-sensitive Ca²⁺-stores.Abbreviations ER endoplasmic reticulum - Ins(1,4,5)P ₃ D-inositol 1,4,5-trisphosphate - Ins(1,3,4)P ₃ D-inositol 1,3,4-trisphosphate - Ins(2,4,5)P ₃ D-inositol 2,4,5-trisphosphate - Ins(1,4)P ₂ D-inositol 1,4-bisphosphate - Ins(1,3,4,5)P ₄ D-inositol 1,3,4,5-tetrakisphosphate - SMC submicrovillar cisternae - [Ca ²⁺]_i intracellular free Ca²⁺-concentration     

Connective tissue activation. XXXIII. Biologically active cleavage products of CTAP-III from human platelets

Evidence for three new isoforms of CTAP-III from human platelets is presented; two NH2-terminal cleavage products were identified, CTAP-III (des 1-13) and CTAP-III (des 1-15). CTAP-III (des 1-13) has a pI of 8.6 and is a relatively stable proteolytic cleavage product that retains the capacity to stimulate [14C]GAG synthesis in human synovial cell cultures. CTAP-III (des 1-15) appears to be an elastase or chymotrypsin cleavage product and identical to NAP-2, an entity thought to have neutrophil activating properties.     

beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22

Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)