Phosphatase activity in the hepatopancreas of Helix aspersa

• 1.1. Phosphatase acid (PhA) activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa has been investigated using cytochemical methods.
• 2.2. All the cells composing this gland show PhA activity, the distribution pattern differing according to the cell type.
• 3.3. The digestive cells show the most widely distributed reaction product (brush border, phagolysosomes, multivesicular bodies and autophagic vacuoles).
• 4.4. In the excretory cells this activity appears in large sacs, while in the calcium cells the reaction product is abundant in the calcium granules.
• 5.5. Cellular digestion processes performed by each of these cell types is discussed together with their role in the detoxification of heavy elements derived from the environment.

     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

Characterization of proteins from mitochondrial ribosomes of Locusta migratoria by three different two-dimensional gel electrophoretic methods and comparison with cytoplasmic ribosomal proteins

• 1.1. Proteins were isolated from subunits of mitochondrial and cytoplasmic ribosomes of Locusta migratoria and were analyzed by means of two-dimensional gel electrophoreses using three different electrophoresis systems.
• 2.2. Using the system of Czempiel et al. (1976) proteins from whole locust mitochondrial ribosomes (combined subunits) were separated into 72 spots; proteins from the large and small subunits resulted in 48 and 29 spots respectively.
• 3.3. The mol. wt distribution of mitochondrial ribosome proteins was estimated by using the electrophoresis system of O'Farrell (1975). These mol. wts are in the range of 11,000–56,000, the average mol. wt is about 29,500. Assuming one copy of protein per ribosome this gives a total mol. wt for the protein part of mitochondrial ribosomes of ca. 2.1 x 10⁶.
• 4.4. Parallel separation of cytoplasmic and mitochondrial ribosome proteins was achieved using the system of Geyl et al. (1981). Cytoplasmic ribosome proteins produced 65 spots and revealed a more alkaline character than mitochondrial ribosome proteins.

     

Pest sequences in nuclear proteins

• 1.1. Most of proteins which are rapidly degraded inside eukaryotic cells have been found to contain amino acid sequences (PEST sequences) enriched in proline, acidic residues (glutamic acid and/or aspartic acid) and hydrophilic residues (serine and threonine) (Rogers et al. (1986) Science234, 364–368).
• 2.2. This correlation was tested on nuclear proteins and a close relationship was found between nuclear protein stability and the presence of PEST regions.
• 3.3. Nuclear proteins with structural functions which can be considered as stable components of cell nuclei generally lack PEST sequences.
• 4.4. In contrast, regulatory nuclear factors which have specific and transient functions generally possess at least one PEST sequence.

     

Seasonal variation in the energy metabolism in an estuarine crab,Chasmagnathus granulata (Dana, 1851)

• 1.1. The effects of seasonal variation on the carbohydrate and lipid metabolism of the Chasmagnathus granulata were investigated.
• 2.2. Glycemia is high in winter and summer and low in spring and fall.
• 3.3. The glycogen content in the hepatopancreas and muscle is higher in fall and winter, and decreases during spring and summer.
• 4.4. The muscle lipids are higher in summer, and decrease during fall and winter whereas hepatopancreas lipids are higher except in the fall.
• 5.5. The crabs show change in the metabolic pattern of lipids and carbohydrates during the seasons of the year.

     

Soluble cyclic amp-dependent protein kinases from chick liver: Effects of NaCl and heat-stable modulator

• 1.1. Two cyclic AMP-dependent protein kinases—Fraction I and II—have been isolated from chick liver soluble preparation on DEAE-cellulose.
• 2.2. Both fractions have an apparent K_m for ATP of 2 × 10⁻⁶M, are stimulated maximally by 5 × 10⁻⁸ M cyclic AMP and phosphorylate mainly basic proteins—histone and protamine.
• 3.3. They exhibit various pH values for optimal activity and show differences with respect to both sensitivity to NaCl and substrate specificity.
• 4.4. The heat-stable protein modulator inhibits the cyclic AMP-dependent protein kinase activity of both fractions, but with cyclic GMP one kinase is stimulated and the other inhibited.
• 5.5. Slight differences in histone triggered holoenzyme dissociation as well as the lack of difference between their ability for subunit reassociation do not allow to classify these isozymes as protein kinases of Type I and II, according to Corbin et al. (1975).

     

Heart rate and its cholinergic control in the sole (Solea vulgaris), acclimatized to different temperatures

• 1.1. The effects of thermal acclimatization at 10 and 24°C on heart rate were investigated on unrestrained soles (Solea vulgaris).
• 2.2. The sensitivity of heart rate to temperature changes induced by temperature acclimatization was higher in cold-acclimatized than in warm-acclimatized soles.
• 3.3. Heart rate of cold-acclimatized fish to temperature changes was not affected by blocking the vagal tone with atropine.
• 4.4. After atropine treatment the ability of heart rate to show thermal compensation decreased in warm-acclimatized soles.
• 5.5. It is suggested that the vagus nerve can function differently at different temperatures.

     

The quaternary structure of the lobster carapace carotenoprotein,crustacyanin: Studies using cross-linking agents

• 1.1. Three bis(imidoesters) of different span (ca 9–11 Å) have been used to form intermolecular cross-links between the apoproteins of the lobster carapace carotenoprotein, α-crustacyanin.
• 2.2. Dimethylpimelimidate(DMP) is the most effective of the three reagents in cross-linking the oligomeric α-crustacyanin, giving predominantly dimers between apoproteins from each of the two apoprotein classes. The native dimers, β-crustacyanins, are effectively cross-linked with this reagent.
• 3.3. The stability of DMP cross-linked α-crustacyanin and of the native carotenoprotein to urea treatment and to heating have been compared.
• 4.4. Reagents of longer (sulpho-N-hydroxy-succinimide ester; 18 Å) or shorter (1,5-difluoro-2,4-dinitrobenzene; 5 Å) spans than the bis(imidoesters) are similarly able to form cross-linked dimers with the crustacyanins, but less effectively under the conditions of the reactions.
• 5.5. The results are discussed in relation to the previously presented putative structure of β-crustacyanin (Keen et al. 1990b. Eur. J. Biochem. (submitted); Zagalsky et al., 1990. Comp. Biochem. Physiol.97B, 1–18) and to an alternative subunit interface arrangement of the apoproteins for the dimer.

     

Mass spectrometric analysis of rat hemoglobin by FAB-overlapping: Primary structure of the α-major and of four β constitutive chains

• 1.1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two α-chain and four β-chains.
• 2.2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the α-major and of the four β-globins.
• 3.3. The primary structure of the α-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J.149, 245–258; Chua et al., 1987).
• 4.4. Sequence analysis of the four β -globin chains showed that amino acid differences are restricted to two protein portions: the region 22–25 and 123–125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22 → Asn in the β₂-globin) involve uncharged substitutions.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Metabolic roles of carnitine expressed through the carnitine acetyltransferase system in Candida pintolopesii

• 1.1. The carnitine-responsive mutant yeast, Candida pintolopesii ATCC 26014 and the wild type strain (ATCC 22987) were used to investigate the role of carnitine and the carnitine acetyltransferase system.
• 2.2. [³H]l-Carnitine, supplied to the cells, was incorporated into acetylcamitine and [¹⁴C]pantothenate was incorporated into CoA and its derivatives.
• 3.3. Both bioautography and quantitative assays indicated that the relative amounts of CoA and acetylCoA were very different in the mutant and wild type cells.
• 4.4. The wild type yeast maintained an acetylCoA/CoA ratio of 0.33 ± 0.09 indicating that most of the CoA in the cell is in the free CoA form. Carnitine was not required to establish this ratio nor did its presence lower it further.
• 5.5. In contrast, the mutant cells contained a high acetylCoA/CoA ratio (12.8 ± 3.0).
• 6.6. In the mutant cells, carnitine lowered the ratio by decreasing the intracellular acetylCoA concentration and releasing free CoA.
• 7.7. These data indicated that wild type yeast possess an effective mechanism that is not related to the CAT system for regulating the acetylCoA/CoA ratio.
• 8.8. This mechanism appears to be lacking in the mutant. The CAT system decreased the acetylCoA/CoA ratio in the mutant cells but not to the value which is found in the wild type strain.
• 9.9. In both stains of Candida pintolopesii, in the presence of carnitine, an acetylcamitine pool can be created whose concentration exceeds that of acetylCoA.
• 10.10. The intracellular apparent equilibrium constant (K_app) for carnitine acetyltransferase for wild type Candida pintolopesii ATCC 22987 was 0.73 ± 0.12, close to the established value of 0.6, indicating that the CAT system ran close to equilibrium.
• 11.11. The K_app for the CAT system of the carnitine-responsive mutant yeast was 7.7 ± 1.7 indicating that this reaction was not at equilibrium.

     

Age-dependent proteins in eyes of annual killifishes Pterolebias longipinnis detected by 2-dimensional electrophoresis

• 1.1. Eye proteins of Pterolebias longipinnis have been analyzed by 2-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis during aging from adolescence until normal death.
• 2.2. The protein pattern on the gels changed gradually with progressing age.
• 3.3. In senescent eyes, three protein spots appeared for a time and 36 disappeared from the pattern.
• 4.4. The isoelectric points of the proteins in the presence of urea and the molecular weights in an unreducing buffer are presented.

     

Optical and neural pooling in visual processing in crustacea

• 1.1. Optical pooling is common in crustacean vision, both pooling in the single ommatidium and pooling inputs from many ommatidia by overlapping visual fields.
• 2.2. Optical and neural pooling work together subdividing the eye into different surface regions with different tasks.
• 3.3. Small-fiber and large-fiber systems with corresponding small and large dendritic branching provide a parallel processing system.
• 4.4. Several parallel, integrating channels process that visual information which is needed for high-speed reactions.
• 5.5. Visual fibers receive contributions from other modality inputs like vibration, olfaction or attention neurons. Inputs from mechanoreceptors transmitted over integrating fibers seem to join the signals in the intergrating visual fibers.
• 6.6. The signal for a particular channel is expressed by the pattern of spikes (rather than changes in the mean frequency of spikes) which is modulated by any input variation.
• 7.7. A particular discharge pattern may then be recognized by a command neuron or a muscle ensemble.

     

Respiration in the eastern water dragon,Physignathus lesueurii (agamidae)

• 1.1. Some aspects of the gas exchange system of a diving lizard, Physignathus lesuewii were studied.
• 2.2. Breathing patterns were analysed.
• 3.3. Breathing rate increases logarithmically with temperature and Q₁₀ = 1.8. LogBR = −0.237 + 0.0256 T.
• 4.4. Gas tensions in lung air and arterial and venous blood were measured. Arterial pH declines with increasing temperature.
• 5.5. Temperature has a marked effect on oxygen affinity of the blood (ΔH = −10.1 kcal mol⁻). A Bohr effect was also noted.
• 6.6. CO₂ equilibrium curves were drawn.
• 7.7. The results are considered with a view to anticipating the efficiency of the gas exchange system of this species under conditions of variable temperature and during diving.

     

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.

     

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.

     

Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.

     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

Differential weighting of electrophoretic data in crayfish and fiddler crabs (decapoda: astacidae and ocypopidae)

• 1.1. Muscle proteins from the chelae of six crayfish species and ten species of Uca were compared through disc electrophoresis (split gel technique).
• 2.2. No intraspecies variation of the electrophoretic pattern was found.
• 3.3. In interspecies comparisons all components (bands) were weighted individually and specified as ancestral or derived characters.
• 4.4. In the crayfishes the phylogenetic trees constructed from electrophoretic and classical data were found to be congruent. In Uca some branches of either tree remained undefined. Each tree, however, helped complete the other one.

     

A halogenated amino acid-containing sperm activating peptide and its related peptides isolated from the egg jelly of sea urchins,Tripneustes gratilla,Pseudoboletia maculata,Strongylocentrotus nudus,Echinometra mathaei and Heterocentrotus mammillatus

• 1.1. Twenty-eight peptides were isolated from the egg jelly of sea urchins, Tripneustes gratilla, Pseudoboletia maculata, Strongylocentrotus nudus, Echinometra mathaei (type A and B) and Heterocentrotus mammillatus and their amino acid sequences were determined.
• 2.2. Two of the peptides obtained from T. gratilla egg jelly possessed a bromophenylalanine (Br-Phe) residue in their sequences (Gly-(Br-Phe)-Asn-Leu-Asn-Gly-Gly-Gly-Val-Gly and Gly-(Br-Phe)-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly).
• 3.3. All of the peptides elevated cyclic GMP concentrations in the spermatozoa of the respective sea urchin and caused a shift in the apparent mol. wt of a major sperm protein of the respective sea urchin.
• 4.4. They stimulated respiration rates of the spermatozoa of Hemicentrotus pulcherrimus as well as their own species.
• 5.5. One-half maximal concentrations of the peptides for respiratory stimulation of H. pulcherrimus spermatozoa were between 10⁻¹¹ M and 10⁻⁹ M except a methionine-containing peptide which was about 10⁻⁷ M.