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.

     

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.

     

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.     

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.

     

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.

     

Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins

• 1.(1) Co-operation between a laboratory interested in developing the theory for protein secondary structure prediction methods and a laboratory interested in applying and comparing such methods has led to the development of a simple predictive algorithm.
• 2.(2) Four-state predictions, in which each residue is unambiguously assigned one conformational state of α-helix, extended chain, reverse turn or coil, predict 49% of residue states correctly (in a sample of 26 proteins) when the overall helix and extended-chain content is not taken into account.
• 3.(3) When the relative abundances of helix, extended chain, reverse turn and coil observed by X-ray crystallography are taken into account, a single constant for each protein and type of conformation can be used to bias the prediction. When predictions are optimized in this way, 63% of all residue states are unambiguously and correctly assigned.
• 4.(4) By analysing the nature of the bias required, proteins can be classified into helix-rich types, pleated-sheet-rich types, and so on. It is shown that, if the type of protein can be determined even approximately by circular dichroism, 57% of residue states can be correctly predicted without taking into account the X-ray structure. Further, comparable predictions can be obtained if, instead of circular dichroism, preliminary predictions are made to assess the protein type.
• 5.(5) It is emphasized that the numbers quoted here depend on the method used to assess accuracy, and the algorithm is shown to be at least as good as, and usually superior to, the reported prediction methods assessed in the same way.
• 6.(6) Ways of further enhancing predictions by the use of additional information from hydrophobic triplets and homologous sequences are also explored. Hydro-phobic triplet information does not significantly improve predictive power and it is concluded that this information is used by proteins in the next stage of folding. On the other hand, the use of homologous sequences appears to be very promising.
• 7.(7) The implication of these results in protein folding is discussed.

     

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.

     

k tibial nerve stimulation: Normative values

Cortical somatosensory evoked potentials to posterior tibial nerve stimulation were obtained in 29 normal controls varying in age and body height. In obtaining these potentials we varied recording derivations and frequency settings. Our recordings demonstrated the following points:

• 1.(1) N20 (dorsal cord potential) and the early cortical components (P2, N2) were the only potentials that were consistently recorded. All other subcortical components (N18, N24, P27, N30) were of relatively low amplitude and not infrequently absent even in normals.
• 2.(2) All absolute latencies other than N2 were correlated with body height. However, interpeak latency differences were independent of body height.
• 3.(3) Below the age of 20, subcortical but not cortical peak latencies correlated with age, but this appeared to be due to changes in body height in this age group.
• 4.(4) Absolute amplitudes and amplitude ratios (left/right and uni/bilateral) showed marked interindividual variability and have very limited value in defining abnormality.
• 5.(5) The use of restricted filter windows facilitated the selective recording of postsynaptic potentials (30–250 Hz) and action potentials (150–1500 Hz).

     

Comprehensive Analysis of the Lysine Succinylome and Protein Co-modifications in Developing Rice Seeds

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Highlights

• •Nonenzymatically Ksu proteins shown different pattern from native cell Ksu proteins.
• •Motif preference of Ksu proteins was associated with different biological processes.
• •Up to 67 developing rice seeds proteins contain PTMs of Kac, Ksu, Kcr, Kmal, and Khib.
• •Some lysine residues of the key pathway enzymes are modified by succinylation.

     

A Combined N-terminomics and Shotgun Proteomics Approach to Investigate the Responses of Human Cells to Rapamycin and Zinc at the Mitochondrial Level

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Highlights

• •Rapamycin and zinc induce moderate but significant mitochondrial proteome changes.
• •The mitochondrial proteins processing system is robust under subtoxic conditions.
• •Rapamycin and zinc perturb the mitochondrial proteins processing system.
• •Rapamycin and zinc perturb the mitochondrial proteins homeostasis.

     

Assembly of the β4-Integrin Interactome Based on Proximal Biotinylation in the Presence and Absence of Heterodimerization,

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Highlights

• •This study reports the first proteomic characterization of a type II hemidesmosomal complex.
• •This study characterizes the interactome of β4-integrin in the presence and absence of α6-integrin in a simple epithelial cell model.
• •The assembly of the β4-integrin interacting complex was largely independent of α6-integrin expression.

     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

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.     

Some physical and chemical properties of purified nematocysts of Hydra attenuata pall. (Hydrozoa,cnidaria)

• 1.1. A method for purifying undischarged nematocysts from Hydra and other cnidarians is described.
• 2.2. Isolated cysts (relative densities 1.22–1.24) evaginate their tubular content even after previous dehydration.
• 3.3. The cyst wall is permeable to dyes of mol. wts up to 600,000.
• 4.4. Approximately two-thirds of the cyst's dry wt are soluble proteins. Eighty per cent of them are of low mol. wt and highly anionic, presumably serving as binding sites for Ca²⁺ and Mg²⁺.
• 5.5. The other 20% includes 30 different proteins amongst them toxins and enzymes (phospholipase and little proteases but no collagenase, chitinase or hyaluronidase).

     

The yolk polypeptides of a free-living rhabditid nematode

• 1.1. The yolk proteins of hermaphrodite Dolichorhabditis sp. (Nematode, Rhabditida) are composed of at least three polypeptides: VT1, VT2 and VT3 with molecular masses of 175.2, 107 and 82 kDa respectively.
• 2.2. All three yolk polypeptides make up at least one native protein complex which can be resolved by PAGE.
• 3.3. The yolk proteins are glycosylated and can be isolated by chromatography in Con A-Sepharose.
• 4.4. Partial chymotryptic hydrolysis shows that VT2 in different from its C. elegans homologue, YP115.
• 5.5. The main polypeptides synthesized by whole animals are the yolk components which are actively secreted in the incubation medium.

     

Identification of a glycoprotein complex containing a glycogen synthase isozyme in Ascaris Suum obliquely-striated muscle

• 1.1. A glycogen/protein complex which contains the major portion of glycogen synthase activity in Ascaris suum muscle has been purified.
• 2.2. The complex contains two proteins which can be dissociated from a glycoprotein component.
• 3.3. The glycoprotein contains glycogen-like domains and is resistant to trypsin digestion.
• 4.4. The glycogen synthase activity in the purified complex catalyzes glycogen synthesis in the absence of exogenous glycogen, but demonstrates an absolute glucose 6-phosphate requirement for activity.
• 5.5. The data support the hypothesis that this isozyme of glycogen synthase is significantly different from the cyclic AMP-regulated enzyme.

     

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

• 1.1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ, grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied.
• 2.2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC.
• 3.3. Individual isoenzymes of one type were separated on FPLC-Mono Q.
• 4.4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C.
• 5.5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

     

Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs

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Highlights

• •Quantitative phosphoproteomics of cells treated with sphingolipid analogs or PP2A inhibitor identify novel protein targets of PP2A.
• •PP2A substrates include several nutrient transporter proteins, GTPase regulators and proteins associated with actin cytoskeletal remodeling.
• •Differential regulation of Akt and Gsk3b account for the difference in vacuolating phenotype observed between SH-BC-893 and C2-ceramide.
• •Dynamic phosphoproteomics enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.

     

Glycolytic enzymes of fowl and turkey spermatozoa

• 1.1. It was confirmed that, under anaerobic conditions, fowl spermatozoa formed lactate from glucose thirteen times faster than turkey spermatozoa.
• 2.2. The profiles of glycolytic enzyme activities were similar for spermatozoa from both species; however fowl spermatozoal activities were generally 2- to 4-fold higher.
• 3.3. Exceptions were glycerophosphate mutase and lactate dehydrogenase activities which were respectively 9.5 and 41 times greater in fowl spermatozoa.
• 4.4. In both species, spermatozoal glyceraldehyde-3-phosphate dehydrogenase had the lowest activity of the glycolytic enzymes.