Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

A Novel Mitochondrial Genome Organization for the Blue Mussel, Mytilus Edulis

The sequence of 13.9 kilobases (kb) of the 17.1-kb mitochondrial genome of Mytilus edulis has been determined, and the arrangement of all genes has been deduced. Mytilus mitochondrial DNA (mtDNA) contains 37 genes, all of which are transcribed from the same DNA strand. The gene content of Mytilus is typically metazoan in that it includes genes for large and small ribosomal RNAs, for a complete set of transfer RNAs and for 12 proteins. The protein genes encode the cytochrome b apoenzyme, cytochrome c oxidase (CO) subunits I-III, NADH dehydrogenase (ND) subunits 1-6 and 4L, and ATP synthetase (ATPase) subunit 6. No gene for ATPase subunit 8 could be found. The reading frames for the ND1, COI, and COIII genes contain long extensions relative to those genes in other metazoan mtDNAs. There are 23 tRNA genes, one more than previously found in any metazoan mtDNA. The additional tRNA appears to specify methionine, making Mytilus mtDNA unique in having two tRNA(Met) genes. Five lengthy unassigned intergenic sequences are present, four of which vary in length from 79 to 119 nucleotides and the largest of which is 1.2 kb. The base compositions of these are unremarkable and do not differ significantly from that of the remainder of the mtDNA. The arrangement of genes in Mytilus mtDNA is remarkably unlike that found in any other known metazoan mtDNA.     

(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin.

The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.     

Nuclear factor I (NF I) binds to an NF I-type site but not to the CCAAT site in the human alpha-globin gene promoter.

Methylation interference and missing contact analyses demonstrate that nuclear factor I (NF I) recognizes an NF I-like site (5'-GGG(N)6GCCAG-3') within the alpha-globin promoter rather than the adjacent CCAAT box. Consistent with this, mutations within the CCAAT box do not alter significantly the affinity and specificity of the interaction whereas elimination of the 5'-GGG-3' half-site of the recognition sequence reduces the DNA binding strength of NF I by 2 orders of magnitude down to the range of unspecific interaction. On the other hand, the mutated alpha-globin promoter sequence that is no longer bound by NF I, although it retains an intact CCAAT box, interacts specifically with a protein component from nuclear extracts of HeLa cells. From these results we conclude that NF I is not the factor that interacts with the CCAAT box and that the second half of the canonical 5'-TGG(N)6GCCAA-3' NF I binding site cannot be regarded as identical with the CCAAT promoter element, as suggested previously.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

A simplified method for the determination of nitric oxide in biological solutions.

A new simplified procedure for determination of nitric oxide (NO) in biological solutions is described utilizing a new reducing system of nitric oxide prior to chemiluminescence. Advantages of the new method makes heating of the reducing solution unnecessary and avoids cooling and condensation of generated vapors. Only traces of acid with a high boiling point are used. The method permits analysis of small sample volumes (200 microL). The basal production of nitric oxide by freshly harvested endothelial cells ranged from 100 to 880 picomoles.     

Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

Sites of ecdysteroid biosynthesis in female adults of Gryllus bimaculatus (Ensifera,Gryllidae)

Summary Ovaries from 4-day-old female adults of Gryllus bimaculatus produce about 5 ng of free and conjugated ecdysteroids per hour during a 16-h incubation in Grace's medium. During incubation of pieces of the abdominal integument together with the adjacent segmental fat body, a net synthesis of moulting hormones is observed (2.3 ng per hour per animal), similar to that in the ovary. Separate incubations of disunited abdominal epidermis and segmental fat body tissue result in much lower rates of ecdysteroid synthesis. Ecdysteroid synthesis in ovarian homogenates is about one-third of that in intact organs. This reduction is due to a lack of conjugate formation in homogenates. Homogenates of the abdominal integument complex are no longer capable of synthesizing ecdysteroids. For both tissues, a de novo synthesis of ecdysteroids is corroborated by following the in vitro incorporation of [¹⁴C]-label from cholesterol and [³H]-label from 2,22,25-trideoxyecdysone (5-ketodiol), respectively, into free ecdysone. The rate of incorporation into ecdysone is only 0.0014% for cholesterol but 0.48% for 5-ketodiol. Both tissues represent primary sources of ecdysteroids in female adult crickets.Abbreviations HPLC high performance liquid chromatography - IU international units - NP normal phase - RIA radioimmunoassay - RP reversed phase - SEM standard error of mean     

Alteration of the Magnetic Properties of Aquaspirillum magnetotacticum by a Pulse Magnetization Technique

The presence of a narrow shape and size distribution for magnetite crystals within magnetotactic organisms suggests strongly that there are species-specific mechanisms that control the process of biomineralization. In order to explore the extent of this control, cultures of Aquaspirillum magnetotacticum in the exponential growth phase were exposed to increasing magnetic pulses with the aim of separating cell populations on the basis of their magnetic coercivities. Isothermal remanent magnetization and anhysteretic remanent magnetization studies were performed with freeze-dried magnetic cells after the remagnetization treatment. Subpopulations of A. magnetotacticum that showed an increase in coercivity correlated with the intensity of the magnetic pulses were isolated. After successive subcultures of the remaining north-seeking cells, a maximum bulk coercivity (H_b^max) of 40 mT was obtained after treatment with a 55-mT pulse. Although we obtained A. magnetotacticum variants displaying higher coercivities than the wild-type strain, changes in crystal size or shape of the magnetite crystals were below reliable detection limits with transmission electron microscopy. Attempts to shift the coercivity towards higher values caused it to decrease, a change which was accompanied by an increase in magnetostatic interactions of the magnetosome chains as well as an increase in the cell population displaying an abnormal distribution of the magnetosome chains. Ultrastructural analyses of cells and magnetosomes revealed the appearance of cystlike bodies which occasionally contained magnetosomes. The increase in cystlike cells and abnormal magnetosome chains when higher magnetic pulses were used suggested that magnetosomes were collapsing because of stronger interparticle magnetostatic forces.     

Phosphofructokinase from mollusc muscle is activated by phosphorylation.

Phosphofructokinase was purified from muscle tissue of two different molluscs, edible snails, Helix pomatia (gastropoda), and mussels, Mytilus edulis (bivalvia). Under denaturing conditions, both enzymes had a molecular mass of 82 kDa. In the presence of ATP-Mg2+, the enzymes were rapidly phosphorylated in vitro by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase purified from snail muscle and also by the C subunit of protein kinase from bovine heart. The extent of phosphorylation was 0.6 and 0.5 phosphate residues per subunit for the snail and the mussel phosphofructokinase, respectively. Phosphorylation of both phosphofructokinases effected a decrease in ATP inhibition at neutral or slightly acidic pH values and increased the affinity for fructose 6-phosphate. The resulting activation in the presence of suboptimum fructose 6-phosphate concentrations was more distinct for the snail enzyme. In addition, phosphorylated phosphofructokinase from mussels exhibited a marked increase in Vmax when activated by either 5'-AMP or fructose 2,6-bisphosphate.