Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary     

Passive immunization protects guinea pigs from lethal toxoplasma infection

Abstract The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti- Toxoplasma immunoglobulins or immune cells that had been enriched for B cells prior to transfer. Cells residing in the spleen, lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 × 10⁷ sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cell-mediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.     

Predator-induced behavioral defense and its ecological consequences for two calanoid copepods

Summary We used an automated technique for the observation and quantification of zooplankton swimming behavior to study the behavioral responses of two congeneric, herbivorous, freshwater copepod prey to a copepod predator (Limnocalanus macrurus). One prey, Diaptomus sicilis, often co-occurs with Limnocalanus, while previous studies indicated that the zoogeographic distribution of the second prey, Diaptomus oregonensis, was independent of the predator. We found that in the presence of Limnocalanus, D. sicilis swims more slowly and with less hopping and jumping than D. oregonensis. Diaptomus sicilis is also attacked and consumed by the predator Limnocalanus macrurus less frequently than D. oregonensis. We suggest that the faster, noisier swimming of D. oregonensis increases its vulnerability to Limnocalanus. The behavioral defenses to both prey are induced by the presence of the predator, and may represent two different anti-predator strategies, crypsis and avoidance for D. sicilis and D. oregonensis respectively. In a zoogeographical analysis D. oregonensis occurs at densities below D. sicilis in lakes where Limnocalanus is at elevated abundances, while in low-predator lakes the opposite is true. This distribution pattern supports our experimental results, and suggests that D. sicilis is adapted to survive with Limnocalanus, while D. oregonensis is not.     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Protein phosphorylation in the facultative chemolithotrophThiobacillus novellus

The phosphorylation of at least five proteins with M_r of about 160,000; 93,000; 85,000; 45,000; and 29,000 respectively was demonstrated in crude extracts from the facultative chemolithotrophThiobacillus novellus. The incorporation of [-³²P]phosphate from ATP into these proteins was dependent on the presence of magnesium ion. The phosphorylation reactions were found to be reversible and required 12.5 mM NaF for maximal activity, indicating the action of phosphatases. In addition, 3,5-cAMP had little effect on protein kinase activity, whereas Ca²⁺ alone was weakly stimulatory. This activation was enhanced by the addition of 3,5-cAMP. Ca²⁺ with calmodulin had a strong stimulatory effect on phosphate incorporation into the proteins. A highly purified preparation containing only the 160, 93, and 85 kDa proteins phosphorylated histone, whereas the uptake of³²P by the three proteins was inhibited. Rabbit muscle phosphorylase b prevented incorporation of radiolabel only into the 160 and 93 kDa proteins.     

Estimation of the stability of globular proteins

The interpretation of ΔG (the free energy change for the reaction, globular conformation ? randomly coiled conformation, in the absence of denaturant), in terms of the free energies of transfer of various parts of the protein molecule from water to denaturant solution, is unsatisfactory because the latter are assumed to be identical to the transfer-free energies of similar groups attached to smaller model compounds. We have made empirical adjustments to transfer-free energy theory that make possible linear extrapolation of the free energy of denaturation of a protein from transition region to zero denaturant concentration. The modified theory, used to analyze the denaturation of proteins by guanidine hydrochloride and urea, allowed us to calculate reasonable values for Δα, the average change in accessibility to solvent of the component groups of protein.     

Effects of superabundant food on breeding success and behavior of the red-winged blackbird

Summary The effects of food on breeding success and behavior of the red-winged blackbird (Icteridae: Agelaius phoeniceus) were investigated during 3 successive breeding seasons. In the second season, a 4-week pulse of abundant food in the form of a periodical cicada emergence (Homoptera: Cicadidae: Magicicada spp.) occurred in the forest adjacent to the marsh where the birds were breeding.During the cicada period, the bird population showed: 1) an increase in foraging trips to the forest and a decrease in trips per h, 2) increased biomass of nestlings, 3) increased nestling survival caused by decreased starvation, 4) increased fledging success, and 5) bimodal weight distributions of older nestlings (reflective of the sexual dimorphism in this species). These data suggest the temporary removal of food limitations on the breeding population when the pulse of food was available.