Monomer-dimer equilibria of a Bence Jones protein and its variable fragment.

The circular dichroic (CD) spectra of a type lambda Bence Jones protein (Tod), its variable (VL) fragment, and the constant (CL) fragment of a type lambda protein (Nag) were measured under various conditions. In the pH region from 5.5 to 7.5, the CD spectra of Tod protein with intact interchain disulfide bond (L(SS)) and and CL did not change with pH, while the spectra of Tod protein in which the interchain disulfide bond had been reduced and alkylated (L(RA)) and VL did not change with pH. The dimerization reactions of L(RA) and VL were studied by following the CD change with protein concentration. The CD spectrum of CL did not change with the protein concentration. The dimerization constant for L(RA) was 4 X 10(4) M-1 at at pH 7.5 and 25 degrees C, which was smaller than that for VL (1 X 10(5) M-1). The ellipticity at 278 nm for the L(RA) dimer was different from that for the L(SS) dimer and changed with pH. These findings indicate that the L(RA) dimer and L(SS) dimer have different conformations. The differences in the conformation and L-L interaction between the L(RA) dimer and L(SS) dimer are discussed on the basis of the conformations of VL and CL and the interactions between the paired domains.     

Resonance Raman study on the active-site structure of a cooperative hemerythrin.

Resonance Raman spectra were observed for the oxy and azidomet forms of a cooperative hemerythrin (Hr) isolated from Lingula unguis and a noncooperative Hr from Siphonosoma cumanense. The O-O stretching frequency of the oxy derivative of the L. unguis Hr was lower in the high-affinity form generated at pH 7.6 than in the low-affinity form generated at pH 6.2, while that of the S. cumanense Hr did not change at those two pH values. The Fe-O-Fe symmetric stretching mode of L. unguis azidomet-Hr exhibited a frequency shift between pH 7.6 and 6.2, while that of S. cumanense was not shifted. However, the corresponding band of the oxy form did not show a pH-dependent frequency change. Therefore, it is noted that the azidomet form is not a suitable model for studying a mechanism of cooperativity, contrary to the structural similarity between the oxy and azidomet forms. The Fe-O2 as well as Fe-N3 stretching frequencies were found to have no relation with the oxygen affinity. Upon exchange of solvent from H2O to D2O, the O-O and Fe-O2 stretching modes of L. unguis Hr were shifted to higher and lower frequencies, respectively, and their magnitudes were the same for the high- and low-affinity forms. The same frequency shifts were observed for S. cumanense Hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light chains of chicken embryonic gizzard myosin.

1. Myosin from gizzards of 15-day-old chicken embryos was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultra-centrifugation and Sepharose 4B chromatography. 2. The myosin composed of heavy and three light chains as determined by sodium dodecyl sulfate (SDS) gel electrophoresis. The molecular weights of the light chains were 23,000 (L23), 20,000 (L20), and 17,000 (L17), respectively. The amount of L23 light chain decreased and disappeared, and the L17 light chain increased steadily in the course of development. The amount of L20 light chain did not change. 3. ATPase activity of the embryonic myosin was essentially the same as that of adult myosin. The change in the light chain pattern in the course of development did not correlate to the ATPase activity. 4. Antigenicity of the heavy chains in the embryonic myosin was the same as that of the adult heavy chains. However, antibodies to light chains were not detected in the antibodies to either the embryonic or adult myosins.     

Learning-induced plasticity in the barrel cortex is disrupted by inhibition of layer 4 somatostatin-containing interneurons

Gaba-ergic neurons are a diverse cell class with extensive influence over cortical processing, but their role in experience-dependent plasticity is not completely understood. Here we addressed the role of cortical somatostatin- (SOM-INs) and vasoactive intestinal polypeptide- (VIP-INs) containing interneurons in a Pavlovian conditioning where stimulation of the vibrissae is used as a conditioned stimulus and tail shock as unconditioned one. This procedure induces a plastic change observed as an enlargement of the cortical functional representation of vibrissae activated during conditioning. Using layer-targeted, cell-selective DREADD transductions, we examined the involvement of SOM-INs and VIP-INs activity in learning-related plastic changes. Under optical recordings, we injected DREADD-expressing vectors into layer IV (L4) barrels or layer II/III (L2/3) areas corresponding to the activated vibrissae. The activity of the interneurons was modulated during all conditioning sessions, and functional 2-deoxyglucose (2DG) maps were obtained 24 h after the last session.In mice with L4 but not L2/3 SOM-INs suppressed during conditioning, the plastic change of whisker representation was absent. The behavioral effect of conditioning was disturbed. Both L4 SOM-INs excitation and L2/3 VIP-INs inhibition during conditioning did not affect the plasticity or the conditioned response.We found the activity of L4 SOM-INs is indispensable in the formation of learning-induced plastic change. We propose that L4 SOM-INs may provide disinhibition by blocking L4 parvalbumin interneurons, allowing a flow of information into upper cortical layers during learning.     

Structural and functional differences of Litopenaeus vannamei crustins

Penaeid crustins were described in Litopenaeus vannamei and L. setiferus as proteins belonging to an antibacterial peptide family with similar sequences but different sizes. Six crustin-coding clones were isolated from a cDNA library from L. vannamei hemocytes, sequenced and compared. Two different isoforms (named I and P) were found, based on two nucleotide differences that produce one change in amino acid sequence (Ile/Pro). Other single differences in nucleotide sequences were also noted, but they did not change the translated product. The mRNA steady state levels of crustin I, but not of crustin P, were down regulated by Vibrio alginolyticus inoculation. Thus, the differences among penaeid crustins seem to be associated with one amino acid substitution, which affects their expression after bacterial inoculation. By structural similarity, shrimp crustins seem to belong to an antibacterial WAP-domain containing protein family.     

Zinc may be a mediator of leptin production in humans

Obese individuals have hyperleptinemia and hypozincemia. Moreover, leptin and zinc have circadian changes in circulating concentrations. We investigated their possible interaction and examined whether a difference existed between obese men and their lean controls. The results indicated the pattern of circadian change in plasma zinc and leptin did not markedly differ between the obese subjects and the lean controls. However, the obese had higher leptin and lower zinc plasma values at each sampling time than did the lean controls. Because an inverse correlation was found in plasma values between zinc and leptin (r=-0.51, p=0.012), we further determined the role zinc might play in leptin production by human subcutaneous adipose tissue from female donors. The in vitro study showed that zinc treatment (0.2 mmol/L) significantly increased leptin production (142%), however, this increment did not surpass that by insulin (10 nmol/L). The data of this study suggest an interactive connection between zinc and leptin.     

The ovary of the wood mouse, Apodemus sylvaticus, during late pregnancy and lactation

Female wood mice, Apodemus sylvaticus, were killed on day 18 of pregnancy (P 18) and on days 0, 3, 6, 9, 12, 15, and 18 of lactation (L 0, L 3, L 6, L 9, L 12, L 15, and L 18 respectively), and the ovaries were studied. The weight of the ovaries was recorded at dissection. The corpora lutea and the follicles of the right ovary were counted and measured, and the appearance of the interstitial tissue was noted. A decline in weight from day P 18 to day L 6 coincided with a decrease in mean diameter of the corpora lutea. The mean number of corpora lutea did, however, not change over the period. The corpora lutea present throughout lactation were probably from the gestation period; the females did not appear to ovulate post-partum. The interstitial tissue was not affected, as far as could be judged with light microscopy. Ovulatory follicles were only present at times close to expected ovulation; on days P 18 and L 18. A lactational anoestrous is suggested for the wood mouse.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

Acute lethal and sublethal effects of neem leaf extract on the neotropical freshwater fish Prochilodus lineatus

The aim of this study was to determine the toxicity of the aqueous extract of neem leaves, a product extensively used in fish-farms as alternative for the control of fish parasites and fish fry predators, for the neotropical fish Prochilodus lineatus. The 24 h LC(50) of neem leaf extract for juveniles P. lineatus was estimated as 4.8 g L(-1); the fish were then exposed for 24 h to 2.5, 5.0 and 7.5 g L(-1) or only clean water (control). Plasma glucose levels were higher in fish exposed to 2.5 g L(-1) and 5.0 g L(-1) neem extract, relative to control, indicating a typical stress response. Neem extract did not interfere with the osmoregulating capacity of the fish, as their plasma sodium, chloride, total protein and osmolarity did not change. The presence of the biopesticide interfered with the antioxidant defense system of P. lineatus, as there was a decrease in liver catalase activity at all neem concentrations and the detoxifying enzyme glutathione-S-transferase was activated in fish exposed to 5.0 g L(-1). Fish exposed to all neem extract concentrations exhibited damaged gill and kidney tissue. These results indicate that although neem extract is less toxic to P. lineatus than other synthetic insecticides used in fish-farming it does cause functional and morphological changes in this fish species.     

Absence of KCNQ1-dependent K+ fluxes in proximal tubular cells of frog kidney

The present study was designed to investigate the functional significance of KCNQ1-mediated K+ secretory fluxes in proximal tubular cells of the frog kidney. To this end, we investigated the effects on rapid depolarization and slow repolarization of the peritubular membrane potential after luminal addition of L-phenylalanine or L-alanine plus/minus KCNQ1 channel blockers. Perfusing the lumen with 10 mmol/L L-phenylalanine plus/minus luminal 293B, a specific blocker of KCNQ1, did not modify the rapid depolarization and the rate of slow repolarization. Perfusing the lumen with 10 mmol/L L-alanine plus/minus luminal HMR-1556, a more potent KCNQ1 channel blocker, did not also alter the rapid depolarization and the rate of slow repolarization. Pretreatment (1 h) of the lumen with HMR-1556 also failed to modify rapid depolarization and rate of slow repolarization upon luminal 10 mmol/L L-alanine. Perfusing the lumen with 1 mmol/L L-alanine plus/minus luminal HMR-1556 did not change the rapid depolarization and the rate of slow repolarization. The pretreatment (1 h) with luminal HMR-1556 did not modify the rapid depolarization and the rate of slow repolarization upon luminal 1 mmol/L L-alanine. The pretreatment (1 h) of the lumen with HMR-1556 did not change transference number for K+ of peritubular cell membrane. Finally, luminal barium blunted the rapid depolarization upon application of luminal 1 mmol/L L-alanine. RT-PCR showed that KCNQ1 mRNA was not expressed in frog kidney. In conclusion, the KCNQ1-dependent K+ secretory fluxes are absent in proximal tubule of frog kidney.     

Identification of an abdominal ganglion neuron antagonizing L7-driven gill contraction in Aplysia

Gill motor neuron L7-induced longitudinal shortening of the gill in Aplysia kurodai and A. juliana was suppressed when extracellular stimuli were applied to a restricted dorsal central region of the abdominal ganglion. We found a neuron there which antagonized the L7-driven contraction. Since the contraction was suppressed when the identified neuron was activated simultaneously with L7, we refer to the newly found neuron as “Anti-L7”. Anti-L7 did not change the L7 impulse generation in the abdominal ganglion. No direct synaptic connection from L7 to Anti-L7 was detected. A fluorescent dye injected into the soma of Anti-L7 revealed that the neuron sent axonal branches to the branchial nerve. These results may show that Anti-L7 antagonizes L7 at the periphery inside the gill, rather than in the abdominal ganglion. EJPs induced by L7 were unaffected by Anti-L7. Activation of Anti-L7 alone did not induce any change in tone or membrane potential of the gill musculature. The suppressive effect of Anti-L7 lasts many seconds after the cessation of a train of Anti-L7 impulses. The results may suggest that the suppression is mediated through an inhibitory neuromodulatory mechanism without inhibition of L7 itself. Accepted: 1 April 1999     

Recovery of maize (Zea mays L.) inbreds and hybrids from chilling stress of various duration: photosynthesis and antioxidant enzymes

The differences between two maize (Zea mays L.) inbred lines and their F1 hybrids in their response to chilling periods of various duration (1, 2, 3 or 4 weeks) and subsequent return to optimum temperatures were analysed by the measurement of the photosystem (PS) 1 and 2 activity, the photosynthetic pigments' content and the activity of antioxidant enzymes. The PS2 activity and the chlorophyll content decreased in plants subjected to 3 or 4 weeks of chilling, but not in those subjected to 1 or 2 weeks of chilling. This decrease was more pronounced in inbreds compared to their hybrids. The activity of superoxide dismutase did not much change with the increasing length of chilling period in the inbreds but decreased in the hybrids, the glutathione reductase activity increased in both types of genotypes but more in the inbred lines, while for ascorbate peroxidase and catalase the changes in parents-hybrids relationship did not show any specific trend. The PS1 activity and the carotenoids' content was not much affected.     

Fluorescence properties of plasma membranes from oats and cauliflower in relation to blue light physiology

Fluorescence properties of plasma membranes from dark-grown oat shoots ( Avena saliva L. cv. Sol II) and from cauliflower inflorescences ( Brassica oleracea L.) were investigated. Along with a flavin (with a possible connection to blue light physiology), a blue fluorescing component was present. The effect of NaN₃, phenyl acetic acid (PAA), KI (flavin inhibitors) and salicylhydroxamic acid (SHAM; inhibitor of e.g. the blue light-induced cytochrome b reduction) were followed with regard to the fluorescence properties of the two components as well as with regard to the light-induced cytochrome b reduction (LIAC). A change in flavin fluorescence and LIAC occurred at about the same concentration of PAA and SHAM, while LIAC was much more sensitive to KI and NaN₃ than was the fluorescence. Rapid freezing and thawing did not change the relative fluorescence emission from the flavin and blue fluorescing component, respectively, but storage at -20°C for one or two days increased the fluorescence, especially from the latter. There did not seem to be a tight coupling between the fluorescence properties of the blue fluorescing component (spectrally similar to a pteridine) and the flavin. Therefore, no conclusions could be drawn concerning their connection in blue light physiology, i.e. in processes such as phototropism.     

The Behavioral Ecology of Libellula luctuosa (Burmeister) (Anisoptera: Libellulidae): I. Temporal Changes in the Population Density and the Effects on Male Territorial Behavior

A single population of a common pond dragonfly, Libellula luctuosa, was studied at a site where the density of males increased dramatically during the breeding season. Early in the summer one active male was found on each territory on the pond. Satellite males were only occasionally found on the territories. Later in the season the number of males per territory increased so that two or more males simultaneously defended on many of the territories, and several satellite males occupied each of the territories. The number and rate of female visitations per day did not change over the summer. These factors resulted in a change in the operational sex ratio with variations in male density. Male behavior was also altered with increasing population density. As male density increased, males were less likely to be seen perching on their territories and more likely to be seen performing aggressive acts such as chasing nearby territorial males and chasing intruders. At high male density, the duration of territorial behaviors was shorter than at low male density. Thus, the percent of a time budget spent in any one activity did not change despite the change in number of males present. Male activity in L. luctuosa is not strictly determined by the opportunity for aggression. Costs of aggression associated with territoriality are minimized by maintaining flexible territorial behaviors.     

Prey selection by juvenile cyprinids from running water

SUMMARY. 1. The juveniles of dace ( Leuciscus leuciscus (L.)) and roach ( Rtuihts rtuihis (L.)) and yearling minnows ( Phoxtnus phoxinus (L.)) exhibited generally similar patterns of feeding behaviour and preferences for particular prey types.
2. Factors affecting prey selection included predator and prey size, prey activity and environmental conditions.
3. All three species preferred prey sizes approximately 0.6x their maximum gape.
4. Prey motion was essential to induce attack, and prey were usually selected in proportion to their relative activities.
5. Specialization on one prey type was observed in still water, and was always directed at the most active prey type. In flowing water no specialization was observed, and size selection was suppressed in the juvenile fish. Yearling minnows, however, were size selective in both still and flowing water.
6. The presence of macrophyte cover did not significantly change the pattern of prey selection, but did reduce the predation rale.
7. These results are discussed in the context of previous studies of prey selection and optimal foraging.     

Nitrogen, phosphorus and light effects on growth and allocation of biomass and nutrients in wild rice

Separating plastic from ontogenetic and growth-limiting responses of plants to changes in resource availability can be challenging because there are a total of eight combinations of these three types of responses. These can, however, be uniquely distinguished on plots of root:shoot ratios against total biomass through time. We used this approach to separate ontogenetic, plastic, and growth-limiting responses of wild rice (Zizania palustris L.) to changes in nitrogen, phosphorus, and light availabilities. Relative growth rate was limited primarily by nitrogen but responded to increased light and phosphorus after nitrogen limitations were alleviated. Nitrogen addition increased relative growth rate because it simultaneously increased unit leaf rate, specific leaf area, and leaf weight ratio. Increased light did not change relative growth rate because decreased specific leaf area and leaf weight ratio compensated the increased unit leaf rate. Phosphorus did not change either relative growth rate or its underlying components. Plants responded ontogenetically to increased nitrogen and light availabilities by accelerating their developmental rate, and plastically by decreasing or increasing their root:shoot ratios, respectively. Plants did not respond either ontogenetically or plastically to increased phosphorus availability. Ontogenetic changes in growth can be separated from plastic and growth-limiting responses by plotting root:shoot ratio against total biomass in the context of the eight possible responses identified above, and also by examining how the underlying components of relative growth rate respond.     

Inactivation of blood plasma alpha 1-proteinase inhibitor as affected by 2 splenic thiol proteinases active in a neutral medium

It has been found that two active in neutral medium thiol proteinases from bovine spleen, cathepsin L and cathepsin H, bring about rapid and irreversible inactivation of alpha 1-proteinase inhibitor (alpha 1PI)--one of the major plasma inhibitors of serine proteinases. The activity of the enzymes studied did not change upon the interaction with alpha 1PI. With stoichiometric proteinase/inhibitor ratio, the inactivation of alpha 1PI under the effect of cathepsin L was instantaneous, while under the effect of cathepsin H it occurred within 30-60 min. The products of alpha 1PI inactivation had an inhibitory effect on the rate of its reaction with cathepsin L. alpha 1PI inactivation under the action of cathepsin L and cathepsin H was accompanied by the decrease in the molecular mass of the inhibitor from 54 kDA to 46 kDa. This was, probably, caused by the hydrolysis of the peptide bond formed by NH2 group of threonine. The 46 kDa fragment did not undergo further degradation. It did not bind to immobilized trypsin but retained antigenic properties. The results obtained show that the limited proteolysis is a mechanism of the inhibitor inactivation. It is suggested that under some conditions thiol proteinases, upon their release from the cell, participate in the control of effective alpha 1PI concentration.