Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

Monoclonal antibodies for radioimmunoscintigraphy of breast cancer

Breast cancer is the leading cause of cancer deaths among females, and it is estimated that each year, one in ten American women will be newly diagnosed as having the disease. It is therefore not surprising, that a great deal of effort has been made to better understand the biology of breast cancer, and that investigators keep up the search for new tools to better characterize, diagnose and treat these tumours. In this regard, the introduction of the hybridoma technique in 1975 by Kohler and Milstein has lead to an extensive work in the characterization of monoclonal and polyclonal antibodies against breast cancers. A large number of antibodies has been raised to different epitopes present in normal and neoplastic breast tissue; but unfortunately we have yet to find a highly sensitive and specific monoclonal antibody for breast cancer that can successfully be used for scintigraphic detection of nodal metastases and for radioimmunotherapy treatment of this disease.As possible radioimmunodiagnostics, antibodies are known which react with the following antigens:

• 1.(1) cytoskeletal proteins
• 2.(2) breast cell products
• 3.(3) steroid receptors
• 4.(4) putative tumor-associated antigens
• 5.(5) oncogene products
• 6.(6) pregnancy-related products
• 7.(7) basement membrane antigens
• 8.(8) degradative enzymes
• 9.(9) cell receptors for extracellular matrix molecules
• 10.(10) multidrug resistance gene product (p-glycoprotein)
• 11.(11) proliferative markers.

     

Accurate Estimation of Context-Dependent False Discovery Rates in Top-Down Proteomics

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Highlights

• •To correctly estimate FDR, search context should be considered.
• •FDR is computed at the molecular level reported; e.g., proteoform or protein.
• •Failure to correctly estimate FDR results in >20-fold errors for the data we studied.
• •TDCD_FDR_Calculator is a free tool providing accurate, conservative FDR estimation.

     

Effects of feeding conditions on toxicity for the purpose of extrapolation

• 1.1. Mathematical models can be most helpful in attempts to understand the way effects of toxic substances show up under various conditions. They are essential for extrapolation and prediction, especially when effects have to be quantified.
• 2.2. In some cases, there is a tight relationship between effects and toxicokinetics, i.e. uptake/elimination behaviour of compounds.
• 3.3. It can be shown that, at least for some compounds, the toxicokinetics depends on feeding conditions. This alone makes it necessary to account for energetics in the use of laboratory observations for predictions and interpretation concerning field data.
• 4.4. It can also be shown that, given certain effects on individuals, the consequences for populations depend sensitively on energetics. This leads to an even tighter link between ecotoxicology and energetics.
• 5.5. A basic problem is that realistic models involve a relatively large number of parameters, even under the most simple assumptions about kinetics and effects. This constraints possibilities for extrapolation and prediction.
• 6.6. All in all, a close link between experimental and modelling programmes is necessary.

     

Functional groups in the social behavior of a cichlid fish,the Oscar,Astronotus ocellatus

Dummy conspecifics were presented to isolated adults of the cichlid fish Astronotus ocellatus to investigate the functional organization of cichlid social behavior. Body size and 15 dummy-elicited activities were recorded during 15 min sessions and analyzed by principal components analysis (PCA) to reveal their temporal organization. Five principal components explained almost 80% of the variation in dummy-elicited behavior, and these five factors define functional groups for

• 1.(a) investigation,
• 2.(b) attack,
• 3.(c) nesting,
• 4.(d) boldness,
• 5.(e)distress.

Nest-oriented and attack modal action patterns are not mutually inhibitory during this time frame, and biting does not appear to function exclusively during an attack on a conspecific. Comparison with previous studies of New and Old World cichlids suggests evolutionary conservation of the functional organization of social behavior.     

Oxidation of methionine residues in equine growth hormone by chloramine-T

• 1.1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone.
• 2.2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved.
• 3.3. Methionine 4 is the most reactive group, followed by methionines 72 and 178—methionine 123 being the less reactive residue.
• 4.4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues.
• 5.5. Results agree with data previously obtained with bovine growth hormone.

     

Isohematoporphyrin-diesters and diethers

• 1.1. A series of diesters of isohematoporphyrin (isoHp), from dimethyl to dioctyl were prepared according to Rimington et al. (1989b). Their optical absorption, fluorescence spectra and high performance liquid chromatography (HPLC) retention times were recorded.
• 2.2. A plot of HPLC retention time against number of C atoms in the alcohol used for esterification was approximately linear at first then rising steeply from diamyl to diocyi ester, whether a gradient elution was used or only methanol: water, 95/5, at pH 7.5.
• 3.3. Preparation of the diethers of isoHp was much more difficult than that of the corresponding derivatives of hematoporphyrin (Hp). Several different methods were investigated, varying both times and temperatures.
• 4.4. These methods included reaction of isoHp or its demethyl ester with
  • 4.1.(i) a bromoalkane in presence of anhydrous K₂CO₃;
  • 4.2.(ii) reaction with bromoalkane and Ag₂O;
  • 4.3.(iii) reaction of brominated-isoHp, prepared by using thionylbromide, with the selected alcohol, or corresponding sodium alcoholate;
  • 4.4.(iv) heating of isoHp alone with an alcohol containing 20% (w/v) H₂SCO₄ (temp. range from 45° to 118°C),
  • 4.5.(v) refluxing as in (iv) at the b.p. of the alcohol; and
  • 4.6.(vi) carrying out this reaction in refluxing ethyleneglycoldimethyl ether (b.p. 85°C) or diethyleneglycoldimethyl ether (b.p. 155°C).
• 5.5. Some diether formation was observable by all these methods but yields were small, a considerable quantity of unreacted isoHp and other products remaining.
• 6.6. Examined by HPLC, the diethers consistently afforded a forked peak which on thin layer chromatography was only resolved into two very closely associated bands by a solvent mixture carefully selected for development.
• 7.7. On elution these materials had virtually identical optical absorption and fluoresence spectra.
• 8.8. The nature of the association is discussed, atropisomers (Gottwald and Ullman, 1969) and possible stacked monomer: dimers (Abraham et al., 1963) being considered as possibilities.

     

The complement system

In summary, the complement system represents a remarkably complex system of interacting proteins noteworthy for several features:

• 1.(1) The assembly and activation of complex proteolytic enzymes, each composed of more than one protein, which act sequentially on specific substrates.
• 2.(2) The biological activity of these enzymes requires the transfer of proteins from the fluid phase to the surface of target cells. This is possible, at least in part, by the ability of C3b and C4b to bind covalently to such surfaces.
• 3.(3) The system is extremely efficient; each of the proteolytically generated fragments appears to perform an important physiological function.
• 4.(4) The final event is the assembly of a macromolecular structure able to penetrate the cell membrane and in so doing lyse the cell.

     

Some effects of dehydration on internal distributions of water and solutes in Xenopus laevis

• 1.1. In relation to body weight changes resulting from evaporative water losses of up to 37% of initial body weight:
  • 1.1.(a) Plasma chloride and potassium concentrations increased in proportion to total body water losses.
  • 1.2.(b) Plasma urea concentrations increased at greater rates than expected from the sum of basal synthesis and dehydration.
  • 1.3.(c) Plasma sodium concentrations initially increased less rapidly than expected from total body water losses, but by losses of 30% of initial body weight closely approximated predicted concentrations.
  • 1.4.(d) Plasma volumes decreased slightly faster than expected, while hematocrits increased as expected.
• 2.2. Skeletal muscles and the ventricular muscles of the heart retained water to greater degrees than expected. Dehydration did not elicit net shifts in Na⁺ K⁺, Cl⁻ or amino acids between the intracellular and extracellular compartments in either skeletal muscle or ventricle.

     

Ecdysone-controlled meiotic reinitiation in oocytes of Locusta migratoria involves a decrease in cAMP levels

Evidence for the involvement of cAMP in the triggering of meiotic reinitiation by ecdysone in vitellogenic oocytes of Locusta migratoria is presented:

• 1.(1) the intracellular concentration of cAMP decreases significantly (by 40%) in the oocytes at the time when meiotic reinitiation is induced;
• 2.(2) drugs which increase the concentration of cAMP antagonize the stimulatory action of ecdysone;
• 3.(3) ecdysone treatment of excised oocytes is followed by a decrease in intra-cellular cAMP;
• 4.(4) ecdysone reduces the adenylate cyclase activity when added to plasma membrane preparations in vitro.

     

Involvement of lysine residue in the nucleotide binding of pigeon liver malic enzyme: modification with affinity label periodate-oxidized nadp

• 1.1. Periodate-oxidized NADP, a competitive inhibitor of malic enzyme with respect to NADP. inactivate the enzyme in mild conditions.
• 2.2. The inactivation is due to the modification of an essential lysine residue.
• 3.3. Two molecules of reagent were found to be incorporated into the enzyme tetramer after extensive modification.
• 4.4. Complete protection of malic enzyme from the oxidized NADP inactivation was afforded by NADP and its analogues.
• 5.5. The modified enzyme showed increased apparent Michaelis constant for the nucleotide coenzymes but the maximum velocity was decreased.
• 6.6. The binding between the modified enzyme and NADPH was impaired.

     

Immunocytochemical localisation of vitellogenic and non-vitellogenic yolk proteins in the ovary of leptinotarsa decemlineata

• 1.1. Two vitellogenins and chromoprotein 2 are selectively accumulated by the oocyte and cannot be detected either in follicle cells or in the germarium.
• 2.2. At the start of their accumulation in terminal oocytes they are asymmetrically distributed.
• 3.3. Endocytosis of vitellogenin 1 starts somewhat later than the uptake of vitellogenin 2 and chromoprotein 2.
• 4.4. In follicle cells of young follicles, a protein (DLP), immunologically related to diapause protein 1, is highly concentrated.
• 5.5. During vitellogenesis DLP is sequestered by the oocytes.
• 6.6. The protein rich globules in terminal oocytes contain the vitellins as well as chromoprotein 2 and DLP.

     

Purification and characterization of the d-lactate dehydrogenase from Leuconostoc lactis

• 1.1. The d-lactate dehydrogenase from Leuconostoc lactis has been purified in high yield.
• 2.2.The enzyme is a dimer of subunits of M_r = 39,000 and each subunit contains a single thiol group. The N-terminal residue is methionine.
• 3.3. The amino acid composition has been determined and is typical of that of a soluble globular protein.

     

Amino acid composition and the protein form of procarboxypeptidase a purified from the pancreas of the sei whale Balaenoptera bolealis

• 1.1. Procarboxypeptidase (W-PCPA) was purified from the pancreas of the sei whale Balaenoptera bolealis.
• 2.2. W-PCPA was obtained as a homogeneous protein in polyacylamide gel disc electrophoresis.
• 3.3. W-PCPA has a molecular weight of 75,000.
• 4.4. Amino acid composition of W-PCPA was compared with that of bovine procarboxypeptidase as A S5 (PCPA-S5).
• 5.5. W-PCPA may be two subunits, and the aggregate form may resemble PCPA-S5.

     

Amino acid substitutions within the analogous nucleotide binding loop (P-loop) of aminoglycoside 3'-phosphotransferase-II

• 1.1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme.
• 2.2. The mutations of Gly205 → Glu, Gly210 → Ala and Arg211 → Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418.
• 3.3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg²⁺ ATP.
• 4.4. Alternatively substitutions at a highly conserved basic residue (Arg211 → Lys and Arg211 → His) were not sufficient for the enzyme to sustain the activity at a level comparable to that of the wildtype.
• 5.5. Moreover, an Arg211 → His mutation drastically reduced affinity of the enzyme for Mg²⁺ ATP.
• 6.6. This argues the importance of Arg211 residue in contributing to the formation of the P-loop structure in addition to its involvement in phosphoryl transfer reaction.
• 7.7. Computer analysis of the secondary structure predicted that the APH(3')-II loop connects a β -strand to an α-helix and that the above mutations caused varying degrees of structural distortions at the corresponding regions of the protein.

     

Phosphorylation of a 25,000-dalton protein in slow and fast muscles of the chicken

• 1.1. Protein phosphorylation in intact chicken latissimus dorsi muscle, slow anterior (ALD) and fast posterior (PLD), was compared.
• 2.2. A major difference in [³²P]phosphate incorporation was found between the ALD and PLD in a 25,000-dalton heat soluble protein.
• 3.3. The 25,000-dalton protein was purified from both the ALD and PLD.
• 4.4. The two proteins had similar amino acid composition and both contained approximately 1 mole phosphate per mole of protein.
• 5.5. The difference in their content of radioactive phosphate was determined to be due to faster turnover in the ALD.

     

The tolerance of lepidopterous larvae to an insect selective neurotoxin

The interaction of the fast-neurotoxic and insect selective polypeptide derived from scorpion venom (AaIT) with lepidopterous larvae tissues was studied through assays of toxicity, chromatography, binding and light microscopical autoradiography. The native and/or radioiodinated toxin was shown to:

• 1.(1) Induce a delayed, slow, progressive paralysis (within 24–48 h) of Spodoptera larvae by relatively high doses (paralytic unit = 2.4 μg/100 mg) corresponding to about only 10% of the total toxicity of the crude venom. Larvae of six species representing five families of Lepidoptera responded similarly to the toxin.
• 2.(2) Resist an in vitro incubation in the insect's hemolymph.
• 3.(3) Lose 80% of its toxicity in the insect's body within 24 h, accompanied by a progressive process of degradation and elimination by the excretory system.
• 4.(4) Specifically bind to a single class of non-interacting binding sites of high affinity and low capacity (0.2 pmol/mg protein, similar to tritiated saxitoxin) in an in vitro, homogenate derived, neuronal preparation.
• 5.(5) Specifically bind with high affinity to desheathed but otherwise intact nerves.
• 6.(6) Be devoid of accessibility to peripheral-terminal branches of Spodoptera motor nerves in situ—strongly contrasting those of the toxin susceptible Periplaneta nerves.

It may be thus concluded that the tolerance of the lepidopterous larvae to AaIT can be substantially attributed to pharmacokinetic aspects of toxin accessibility barriers and degradation processes.     

Evidence of presence of a low molecular weight,non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

• 1.1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments.
• 2.2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt.
• 3.3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine.
• 4.4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt.
• 5.5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated.
• 6.6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Domain structure of the 90-kDa stress protein: Heparin- and antibody-binding domain

• 1.1. To understand the physiological roles of the 90-kDa stress protein (HSP90), we investigated the heparin- and antibody-binding domains of the protein.
• 2.2. For heparin-binding sites, HSP90 was digested completely with trypsin, and the digests were applied to a heparin-Sepharose column and eluted with 1.0 M NaCl, followed by 8.0 M urea.
• 3.3. Each elutant was purified by a reverse-phase C₁₈ column.
• 4.4. Two peptides from the NaCl-eluted fraction and no peptide from the urea-eluted fraction were purified.
• 5.5. The purified peptides were sequenced by an automated peptide sequencer.
• 6.6. One of the heparin-binding sites was present between Leu-362 and Arg-365; another was present between Leu-645 and Lys-648.
• 7.7. These two peptides were basic and considerably hydrophilic.
• 8.8. For antibody-binding sites, HSP90 was mildly digested with trypsin, electrophoresed on SDS-polyacrylamide gels and transferred to PVDF membranes.
• 9.9. The four bound of the trypsin fragments could be sequenced with a peptide sequencer.
• 10.10. There was only one antibody-binding peptide, 38 kDa, starting from Pro-2. The others showed no cross-reactivity with the antibody and started from Leu-283.
• 11.11. Therefore, the epitopes of HSP90 are present between Pro-2 and Leu-282.
• 12.12. The heparin-binding sites are present from the middle region of the HSP90 molecule, and the antigen sites are at the N-terminal domain.