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.

     

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.

     

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.     

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.

     

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 effect of eyestalk ablation on growth,haemolymph composition and gill Na+, K+-ATPase activity of Penaeus monodon juveniles

• 1.1. Eyestalk unablated and unilaterally ablated Penaeus monodon juveniles had survival rates after 5 months of 75–72.5 and 67.5–60%, respectively.
• 2.2. Unilaterally ablated shrimps had significantly higher (P < 0.05) growth rate than unablated shrimps.
• 3.3. Eyestalk-ablatement resulted in a decrease in the haemolymph sodium concentration and an increase in the potassium and calcium concentration of shrimps.
• 4.4. The osmolarity of haemolymph and total protein concentration of unablated shrimps were demonstrated to be higher than those of unilaterally ablated shrimps.
• 5.5. The eyestalk-ablated shrimps possess higher total ATPase and Na⁺,K⁺-ATPase activities in the gill than those of unablated shrimps.

     

Thermal properties for digestive enzymes of a sub-antarctic beetle,hydromedion sparsutum (coleoptera,perimylopidae) compared to those in two thermophilic insects

• 1.1. Proteolytic, lipolytic, amylolytic and cellulolytic activities were studied in adults of the phytophagous beetle, Hydromedion sparsutum, indigenous to the sub-Antarctic island of South Georgia.
• 2.2. Gastric enzyme activities were measured at experimental temperatures of 5–40°C and results were compared with those obtained from two thermophilic insects, Gryllus bimaculatus and Tenebrio molitor.
• 3.3. Protease and lipase activities in Hydromedion were 10–15 times lower than in Gryllus and Tenebrio.
• 4.4. In the temperature range of 5–15°C, α-amylase activity from Hydromedion was only slightly lower than that from Gryllus.
• 5.5. Hydromedion gut homogenates exhibited a distinct cellulolytic activity, even at a low temperature of 5°C.
• 6.6. Cellulolytic activity in the digestive tract of Hydromedion was confirmed by the evolution of ¹⁴CO₂ after consumption of labelled cellulose.
• 7.7. The thermal properties of digestive enzymes agree well with the role of Hydromedion as primary decomposer in its ecosystem.

     

The inhibitory effect of tetramethylethylene diamine on water soluble and membrane bound acetylcholinesterase activity

• 1.1. The inhibitory effect of N,N,N′,N′-tetramethylethylene diamine (TEMED) on water soluble (WSAChE) and membrane bound (MBAChE) acetylcholinesterase was investigated.
• 2.2. TEMED (0.5–4.0 mM) reversibly inhibited WSAChE activity (18–62%) and MBAChE (20–61%) in a concentration dependent manner.
• 3.3. The IC₅₀ being about 2.8 mM for WSAChE and 2.6 mM for MBAChE.
• 4.4. Lineweaver-Burk plots indicated that the nature of inhibition is noncompetitive for both water soluble and membrane bound acetylcholinesterase, with K_m values 68 μM and 123 μM respectively.
• 5.5. An Arrhenius plot showed that the transition temperature (TT) is unaffected in the presence of TEMED.
• 6.6. The activation energy was increased below and above TT in the case of WSAChE only.
• 7.7. On the basis of this behaviour of TEMED with AChE. it can be proposed that it can be used as an eluting agent for the bounded AChE to affinity ligand and may have beneficial action on the reactivatability of irreversibly-inhibited AChE due to its structure.
• 8.8. Moreover there is a possibility that it can be used as a therapeutic agent for the treatment of Alzheimer's disease, myasthenia gravia and glaucoma like some other inhibitors of AChE.

     

The digestive enzymes of the pacific brown shrimp Penaeus californiensis.: I—Properties of amylase activity in the digestive tract

• 1.1. Crude extract of the whole digestive tract from the brown shrimp (P. californiensis) was investigated for digestive amylase activity.
• 2.2. Considerable amylase activity was found at pH 6.5–8.0, with optimum pH at around 7.5.
• 3.3. Optimum temperature was found between 30–40°C, similar to amylases from other crustaceans.
• 4.4. Amylase activity was highly halotolerant, having 50% maximum activity at 3 M NaCl.
• 5.5. Maximum amylase activity was found at 0.01 M NaCl.
• 6.6. Amylase activity was partially inhibited by the divalent ions Hg²⁺, Zn²⁺, Cu²⁺ and Cr²⁺.
• 7.7. Mg²⁺ and Ca²⁺ ions seemed to enhance amylase activity.

     

Effects on KLa of hole diameter and free cross-sectional area of perforated plates in a gas-stirred reactor

The effects on the K_La value of hole diameter and the ratio of free cross-sectional area of perforated plates were studied by using a gas-stirred reactor which had five short draft tubes, two of which were covered with perforated plates and the rest were free.Results showed that the superficial gas velocity is the only factor affecting the K_La value, if the following three conditions are satisfied.

• 1.(1) The superficial gas velocity is higher than 4 Ncm/s.
• 2.(2) The diameter of each hole in a perforated plate is larger than 5 mm.
• 3.(3) The free cross-sectional area exceeds 5%.

     

Stiffness of starfish arm and involvement of catch connective tissue in the stiffness change

• 1.1. Bending tests were performed on intact arms of two starfishes of different orders to obtain stiffness of the arm.
• 2.2. Linckia laevigata without stimulation showed a wide variety of arm stiffness of 2.24–51.3 MPa (average: 8.0 MPa). Mechanical stimulation increased the stiffness by 2.5 times.
• 3.3. In Asterias forbesii, isolated arms were 23 times stiffer than intact ones. Anesthesia with 0.1% MS-222 or menthol-saturated sea water increased the stiffness by 7–170 times.
• 4.4. Ion dependence of stiffness suggests that the catch connective tissue was involved in the stiffness change.

     

Comparative study of proteolytic enzymes in the digestive tracts of the european sea bass and hybrid striped bass reared in freshwater

• 1.1. A comparative study of the proteolytic activity in four different sections of the digestive tracts of the European sea bass (Dicentrarchus labrax) and hybrid striped bass (Morone chrysops × M. saxatilis) reared in freshwater revealed minor differences between these fish.
• 2.2. Tryptic activity plays a major role in the proteolytic process in both fish.
• 3.3. The activity of seven intestinal proteolytic enzymes was detected utilizing a combination of specific substrates and inhibitors.
• 4.4. High levels of proteolytic activity were detected in both the proximal and distal sections of the fish intestine at a high pH range (9–10).
• 5.5. In situ monitoring of pH levels revealed a lower pH level in the intestinal proximal section of hybrid striped bass compared with the distal section.
• 6.6. In contrast, higher pH levels were detected at the proximal compared with the distal sections of D. labrax intestine.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Insulin,glucagon and catecholamine interactions in avian liver explants

• 1.1. A mechanical tissue chopper was used to obtain liver explants (35–75 mg) from 2- to 3-week-old chickens to determine both tissue sensitivity and metabolic effects of isoproterenol, avian insulin and glucagon.
• 2.2. Avian insulin had no effect on lipogenesis; however, lipogenesis was decreased by dibutyryl cyclic AMP. Insulin did not overcome a decrease in lipogenesis caused by catecholamines. Therefore, this control mechanisms is not modulated by insulin.
• 3.3. Preincubation in the presence of glucagon decreased in vitro lipogenesis. Preincubation in the presence of a 19–29 amino acid construct that approximated the radioimmune site for glucagon did not result in a similar effect. Therefore, this site does not relate to the biopotency of the hormone.
• 4.4. A previously noted catecholamine induced decrease in in vitro lipogenesis was verified, showing that points of in vitro regulation are under phosphorylation-dephosphorylation control.
• 5.5. Preincubation of slices (1 hr) with propranolol blocked the inhibition of lipogenesis caused by α and β adrenergic agonists (arterenol or isoproterenol) during a subsequent 2-hr incubation.
• 6.6. Preincubation of slices with either of these agonists decreased lipogenesis even following an extensive washout.
• 7.7. Inhibition could be overcome with propranolol, a β adrenergic antagonist.

     

Cellular and intracellular localization of catalase and acid phosphatase in the midgut of Lumbricus terrestris L.: A cell fractionation study

• 1.1. Cellular and intracellular localization of catalase and acid phosphomonoesterase in the midgut of Lumbricus terrestris was studied by use of tissue fractionation.
• 2.2. At least 60–70% of the catalase resides in the chloragocyte cytosol and the remaining 30–40% resides in gut epithelium peroxisomes.
• 3.3. One of the main functions of the chloragocyte catalase is probably scavenging for H₂O₂ arising from the interaction between blood heme-protein and oxygen.
• 4.4. A simple method for the histochemical detection of cytosol catalase is proposed.
• 5.5. About 10% of the gut acid phosphatase resides in chloragocyte lysosomes. The chloragosomes contain no acid phosphatase.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Changes in composition and proteolytic enzyme activities of artemia during early development

• 1.1. The proximate composition, total and free amino acids, and proteases of Artemia nauplii were determined during early development.
• 2.2. Moisture increased from 71.0% to 80.8%, crude protein decreased from 13.2% to 8.8%, crude fat and ash varied slightly.
• 3.3. The total amino acids decreased. Free amino acids changed in three patterns.
• 4.4. Trypsin, chymotrypsin, carboxypeptidase A, B and cathepsin B and C increased in activity. The activity of trypsin was lower, while cathepsin B and C were the highest.
• 5.5. The protease activities were maximal at pH 7.5 and 8.0, and at 45°C on casein.
• 6.6. The optimal pH for carboxypeptidase A was 4.0, for carboxypeptidase B was 4.5, for trypsin and chymotrypsin were 7.0–7.5. The protease(s) active at pH 9.0–9.5 were to be determined.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.