The effect of nematode parasitism on the osmolality and major cation concentration in the haemolymph of three larval mosquito species

Parasitism by a mermithid nematode, Romanomermis culicivorax, causes severe depletion of haemolymph carbohydrates and proteins in mosquito larvae. We undertook a study to determine if haemolymph osmolality and cation concentrations were affected also by mermithid parasitism. The haemolymph osmolality of R. culicivorax-infected and control Aedes taeniorhynchus and Culex pipiens fourth-instar larvae was not significantly different. However, the haemolymph osmolality decreased significantly in infected Anopheles quadrimaculatus. Each mosquito species demonstrated significant alterations in the haemolymph concentration of at least one cation when infected although the cation concentrations affected differed for each species. The changes observed were statistically significant but the magnitude of change was not great. Overall, despite the severe nutritional burden of the mermithid nematode, these species of mosquito larvae can continue to maintain osmoregulation.     

Assembly of cyanobacterial and higher plant ribulose bisphosphate carboxylase subunits into functional homologous and heterologous enzyme molecules in Escherichia coli

The genes for the large (rbcL) and small (rbcS) subunits of ribulose-1,5 bisphosphate carboxylase-oxygenase (RuBPCase) from the cyanobacterium Synechococcus PCC 6301, and the rbcS gene of wheat, have been expressed in Escherichia coli in order to study homologous and heterologous enzyme assembly. Synechococcus L subunits expressed in E. coli in the absence of S subunits assemble into oligomeric structures without detectable enzyme activity. Co-expression of L and S subunits, achieved after infection with an M13 recombinant phage containing the rbcS gene, restores enzyme activity, thus demonstrating the essential role of S in the formation of an active RuBPCase. The S subunit, however, is neither required for the solubility nor for the assembly of the L subunits into oligomeric forms. The specific activity of the homologous Synechococcus RuBPCase can be modulated by changing the intracellular pool size of S by phage infection. Heterologous assembly between L subunits of Synechococcus and S subunits of wheat can be demonstrated and results in a functional enzyme. The hybrid RuBPCase has approximately 10% of the activity of the homologous Synechococcus enzyme.     

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man

Summary Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose(1 4)-N-acetylglucosamine (Gal(1 )-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal -Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(1 3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal -Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose.The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize P_o protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.This research was supported by NIH Grants AM-10956, HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant No. 79     

Identification of proapoa-I in rat lymph and plasma: metabolic conversion to "mature" apoA-I

Rat apoA-I polymorphism has been analyzed in lymph and plasma. Two major proteins were present and their relative distribution was different in lymph and plasma lipoproteins. The basic protein (pI 5.60) was quantitatively most abundant among plasma lipoproteins and the acidic protein (pI 5.50) was predominant in lymph chylomicrons and lipoproteins. Microsequence amino acid analysis of the two proteins isolated by preparative isoelectrofocusing revealed that pI 5.50 apoA-I was proapoA-I with six additional amino acids (H2N-Ser-Glu-Phe-Trp-Gln-Gln) at the N-terminal end of "mature" apoA-I (pI 5.60 apoA-I). When radioiodinated proapoA-I was injected in rats, a conversion to "mature" apoA-I was observed and the process reached 92% completion in six hours. These data demonstrate the origin of apoA-I polymorphism in vivo.     

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.     

Mechanism of mutation at the aprt locus in Chinese hamster ovary cells: analysis of heterozygotes and hemizygotes.

A two-step model to explain the high frequency of mutation at the diploid adenine phosphoribosyltransferase (aprt) locus in CHO cells has been proposed previously (Simon et al., Mol. Cell. Biol. 2:1126-1133, 1982). This model indicates that two distinct classes of aprt heterozygotes can be isolated. Class 1 heterozygotes, the most abundant class, were defined as those which arose spontaneously and were capable of undergoing mutation to the APRT- phenotype only at a low frequency (putative point mutation). Class 2 heterozygotes arose from a mutation and gave rise at a high frequency to APRT- cells. This high-frequency event has been identified as a deletion of the wild-type allele (A. E. Simon and M. W. Taylor, Proc. Natl. Acad. Sci. U.S.A. 80:810-814, 1983). In this paper we report further analysis of class 1 heterozygotes with respect to genetic structure, gene products, and karyotype. Our study indicated that class 1 heterozygotes contain two different types of mutants. About half have only one copy of the aprt gene and an unaltered karyotype, indicating that a deletion (similar to the high-frequency second-step event observed for class 2 heterozygotes) rather than a loss of the chromosome was responsible for the generation of the aprt+/- genotype. The remainder of the previously designated class 1 heterozygotes still contained two copies of the aprt gene (within the limits of the quantitation technique used) and arose presumably by a point mutation. One of this group, D423, was characterized with respect to aprt gene products and found to produce an electrophoretic variant in addition to the wild-type protein. APRT- mutants derived from D423 retained the same number of aprt gene copies as D423 and still synthesized a protein that comigrated with wild type, unlike APRT- mutants derived from class 2 heterozygotes. D423 and the other heterozygotes with two aprt genes therefore did not fit into either class 1 or 2 and are now designated class 3. The model we present suggests that only one of the two aprt alleles present in wild-type cells can undergo the deletion.     

Studies of the interaction of RecA protein with DNA

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.     

Regulation of Substrate Uptake in Nocardia erythropolis

The regulation of uptake of glucose (GLU), glycerol (GLY), mannitol (MTL), and succinate (SUC) has been examined in Nocardia erythropolis 305. The apparent K_m values of the uptake activities of cells subcultured in a medium with the corresponding substrate as the sole carbon source were 205, 48, 8.7, and 36 μM for GLU, GLY, MTL, and SUC, respectively. GLU and GLY uptake activities were constitutive, although there was evidence for an additional inducible component in GLY uptake. Moreover, MTL and SUC uptake activities were inducible. MTL uptake activity was markedly induced by cultivation in MTL medium and partially induced by growth in GLU medium, whereas SUC uptake was induced only by cultivation in SUC medium. SUC added to MTL medium partially repressed the formation of, or inhibited the activity of, MTL uptake. When not induced, uptake of MTL and SUC was proportional to the substrate concentration. The induced uptake of MTL and SUC and the constitutive uptake of GLU were energy dependent and carrier mediated. Uptake of GLY, constitutively or when induced, was also carrier mediated.     

Some observations of Octopus joubini reared in an inland aquarium

Octopus joubini Robson were hatched and reared in a closed circulating sea-water system. Mortality was greatest during the period of hatching. The longest surviving octopus lived for 128 days—it had increased its weight × 5, from 0·04–0·19 g, mantle length × 3, from 3–9 mm, head width × 2, from 3–5 mm. Observations of (1) general behaviour, (2) feeding behaviour, (3) social behaviour of the newly hatched octopuses (0–3 days) and the three longest surviving octopuses (93–119 days) are described. In particular it was observed that the older octopuses would readily grasp and eat small live crabs when these were made to touch the suckers or arms. On no occasion did the attacks appear to be visually elicited. This result is discussed with reference to the memory and learning experiments using O. vulgaris. Possible reasons for the limited survival are also discussed.