An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin <Emphasis Type="Italic">Anthocidaris crassispina</Emphasis>

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles (zymogen granules) and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in empty vacuoles inside the remnant body. The empty vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of cheese-hole-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.We are grateful to the Research Committee, City University of Hong Kong, for the award of a Strategic Grant (no. 7000989). This work also was supported by a grant from Minpromnayka NS 1219.2003.4     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

The level of free amino acids in erythrocytes of different breeds of hen

Summary The content of free amino acids was determined in erythrocytes of adult Leghorn (Lg, White Rock (WR) and Cornish (Cr) hens, bred under identical conditions. The concentration of total amino acids was twice as high in the erythrocytes as in plasma, amounting to 396 m/100 ml, 424 m/100 ml and 475m/100 ml in White Rock, Cornish and Leghorn hens, respectively.Significant differences were found in the ratio of basic amino acids to acidic amino acids. These values were 0.76, 1.75 and 3.19 in White Rick, Leghorn and Cornish hens, respectively; in the plasma of all 3 breeds the ratio was 1. Statistically significant interbreed differences were expressed more distinctly in erythrocyte than in plasma amino acid concentrations. For absolute concentrations the differences were significant in the case of 9 amino acids.     

Inactivation of voltage-dependent calcium current in an insulinoma cell line

We have studied the mechanism of Ca current inactivation in the -cell line HIT-T15 by conventional and perforated patch recording techniques, using two pulse voltage protocols and a combination of current and tail current measurements. In 5 mM Ca, from a holding potential of - 80 mV, the maximum current showed a complex time course of inactivation: a relatively fast, double exponential inactivation (_h1 12 ms and _h2 60 ms) and a very slowly inactivating component ( > 1 s). The faster component (_h1) was due to the voltage-dependent inactivation of a low-threshold-activated (LVA), T-type current, which deactivates more slowly ( 3–5 ms) than the other components ( 0.2–0.3 ms). The intermediate component (_h2) was due to the Ca-dependent inactivation of a portion of the high-threshold-activated (HVA) current. A saturating dose of the dihydropyridine (DHP) nifedipine (10 M) did not affect the LVA current, but inhibited by 68 ± 5% the transient, Ca-sensitive portion of the HVA current and by 33 ± 12% the long lasting component. We suggest that three components of the calcium current can be resolved in HIT cells and the main target of DHPs is a HVA current, which inactivates faster than the DHP-resistant HVA component and does so primarily through calcium influx. Correspondence to: C. Marchetti     

Mutation induction by γ and X-ray irradiation in tissue cultured lotus

Mutations of tissue cultured lotus were induced by treating plantlets with either acute -rays at doses of 0, 2, 3, 4, 5 or 6 krad or X-rays at doses of 0, 1, 2, 3, 4 or 5 krad. The 2-krad dose of either - or X-ray treatments resulted in a 50% survival rate. The use of - and X-rays to induce mutation in lotus resulted in 21 altered characteristics. Mutants from 1- and 2-krad of either or X-rays had long secondary roots and numerous adventitious roots. These mutants also exhibited good shoot growth and healthy rhizome development. Most plants treated with 3–5 krad of either - or X-rays exhibited abnormal characteristics including vitrification, chlorosis, deformed petioles and in addition had inhibited growth of lateral buds, secondary roots and rhizomes. All plants treated with 6 krad of -rays died within 4 weeks. Control plants had stoma lengths of 2.56 m and cytological analysis of the root tips confirmed the diploid chromosome number of 16. Two groups of aneuploid cells were achieved using irradiation at doses of 3 and 4 krad of either - or X-ray. Chromosome numbers were 2n=18 and 20 with associated stoma lengths of 3.43 and 4.34 m, respectively. Abnormal stomata (cyclocytic and deformity) were observed in plants treated with 4 krad of -ray.     

Essential role of the small subunit of thermostable glucose dehydrogenase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The co-expression in Escherichia coli of the -subunit and the catalytic -subunit of the thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 produced 12.7 U GDH activity mg^–1 protein. A 47-amino acid, twin-arginine translocase signal peptide was identified at the amino terminus of the -subunit. The expression of the -subunit in the absence of the -subunit or the -subunit signal peptide failed to produce any detectable GDH protein or activity. The -subunit may be a chaperone-like component that assists folding of the -subunit polypeptide to the active form and its translocation to the periplasm.     

Dicentric chromosomes in monolayers of human lymphocytes produced by monochromatized synchrotron radiation with photon energies from 1.83 keV to 17.4 keV

A systematic approach was used to examine the induction of dicentric chromosomes in human lymphocytes irradiated in vitro with monochromatized synchrotron radiation produced with photon energies in the range from 1.83 keV to 17.4 keV. To avoid potential confounding factors that could influence the outcome of the experiments, only blood from one individual was used. Since for the irradiation experiments with these low photon energies the local dose variations would become unacceptable, monolayers of lymphocytes attached within 3 h PHA stimulation have been used. The culture conditions ensured that the chromosome analysis could be performed exclusively in metaphases of the first cell cycle in vitro. There is a strong indication for a systematic change of the coefficient of the linear quadratic dose-response relationship from 1.83 keV (=1.26±0.28) with increasing energy up to 6.9 keV (=8.24±0.41) and a decrease with further increase of energy up to 17.4 keV (=3.83±1.72). A tendency for a systematic change of the coefficient seems to be present at energies of 6.9 keV (=8.04±0.40) and 4.8 keV (=9.48±1.57) as well as at energies of 3.10 keV (=2.99±0.51) and 1.83 keV (=0.40±0.25). These results agree in essence with the previously published large data set from Sasakis laboratory.     

Cyclodextrins as a useful tool for bioconversions in plant cell biotechnology

The application of cyclodextrins as precursor solubilizers in biotechnological processes, in which plant cells are involved, is new. In this paper the possibilities for cyclodextrin facilitated bioconversions by freely suspended and/or immobilized plant cells or plant enzymes are demonstrated. After complexation with -cyclodextrin, the phenolic steroid 17-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with -cyclodextrin the solubility of the steroid increased from almost insoluble to 660 M. In addition, by complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed to cell cultures of Podophyllum hexandrum in order to enhance the accumulation of podophyllotoxin. Finally, the glucosylation of podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: -cyclodextrin, -cyclodextrin, hydroxypropyl--cyclodextrin and dimethyl--cyclodextrin were used to improve the solubility of podophyllotoxin. Dimethyl--cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM. Podophyllotoxin--d-glucoside was formed at a rate of 0.51 mmol l^-1 suspension per day by the L. flavum cells growing in the presence of 1.35 mM podophyllotoxin, complexed with dimethyl--cyclodextrin.Abbreviations DW dry weight - E2 17-estradiol - FW fresh weight - PCV packed cell volume     

Degradation of alpha, beta, gamma and delta-hexachlorocyclohexanes by Sphingomonas paucimobilis

Sphingomonas paucimobilis degrades aerobically , , and -hexachlorocyclohexane. With -HCH, complete degradation occurred after 3 days but with and , and with -HCH, 98 and 56 % degradation occurred after 12 and 8 days of incubation, respectively. Pentachlorocyclohexene was formed as the primary metabolite during the degradation of all the HCH isomers. © Rapid Science Ltd. 1998     

On age morphological changes of males of Chydoridae (Cladocera)

Summary Young and adult males of 11 species of Chydoridae are studied, their figures being published here (fig. 1–15). The necessity is stressed to distinguish young forms of males and gynandromorphic individuals.Pleuroxus balatonicus is considered to be described from the population ofPleuroxus unicatus having under Balaton Lake conditions retarded transformation of young males into adult form, and accordingly having unusually numerous young males. \qO\qs\qn\qo\qv\qn\qy\ye \qr\ye\qz\qu\ql\Qj\qt\qa\qt\qy 11 (. 1–15). . , Pleuroxus uncinatus , Pleuroxus balatonicus.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Oligo-riboprobes

Summary In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. More-over, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes (oligo-riboprobes). These probes can be labelled to very high (10⁹ cpm/g) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from preprotachykinin cDNA.     

Mass spectrometric analysis of genetic and post-translational heterogeneity in the lectins Jacalin andMaclura pomifera agglutinin

Jacalin andM. pomifera agglutinin are T-antigen specific lectins with ₄₄ structures that show far greater microheterogeneity than plant lectins from other families, due to multiple genetic isoforms and post-translational processing. Electrospray mass spectrometry and combined liquid chromatography-electrospray mass spectrometry were used to characterize the various forms. For both lectins, the mass data were consistent with previous protein sequencing of the major -chain species of 133 residues and three -chain species of 20 or 21 residues. In addition, for jacalin the mass of one minor -chain species was consistent with a second of the four reported gene sequences. However, the glycopeptide -chain form and one -chain form did not match any of the genes, suggesting a fifth gene remains to be found. ForM. pomifera agglutinin, three more -chain forms were found, but all six could arise from only two genes, with additional post-translational proteolysis and post-translational substitution with an unidentified component of 106 Da creating the set of six forms. Only two -chain forms were found also, with no glycosylation.     

Morphological development and gibberellin production by different strains of Gibberella fujikuroi in shake flasks and bioreactor

Strain H-984 of G. fujikuroi grown for 38h in a shake flask with medium containing 20g glucose l^–1, 3g yeast extract l^–1, 2.5g NH4NO3 l^–1, 0.5g KH2PO4 l^–1, 0.1g MgSO4 l^–1, 1g CaCO₃ l^–1, and inoculated into a bioreactor with medium containing 60g glucose l^–1; 1g NH_4Cl l^–1; 3g KH2PO4 l^–1 and 1.5g MgSO₄ l^–1 produced 1100mg gibberellic acid l–1.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

A reinterpretation of Na channel gating and permeation in terms of a phase transition between a transmembrane S4 α-helix and a channel-helix

A functional model for the S4/IV -helix of the action potential sodium channel is described by means of a thermodynamic approach. The model is based on a phase transition between the -helix and an ion conducting channel-helix which is similar to the well established helix-coil transition in solution. The right hand channel-helix is a peptide chain with an alternating sequence of torsional angles (¹¹)=(87°, 315°) and (²²)=(22°, 107°) which yields a helix of 13.5 Å per turn. The axial dipole moments of the peptide bonds of this chain of l-amino acids nearly cancel each other out in similar way to those in the gramicidin A channel, which is formed by alternating d-and l-amino acids. The helix, which does not contain any H-bonds, is stabilized by a helical file of water molecules which includes the permeating ion(s). This file turns around the channel-helix to form a relatively stable double helix structure which corresponds to the open channel. Since every third side chain in the S4/IV helix carries a positive charge their environments must be polarized. These polarized regions form a left hand screening-helix around the -helix are broken and the internal -carbon atom is considered as fixed, the outer ten residues leave the membrane while the internal ten residues form the channel-helix. In this configuration every positively charged side chain matches nearly exactly every second polarized region of the screening-helix leaving the three regions in-between exposed to the water file containing the ion(s). This further stabilizes the channel and agrees nicely with the idea of cationic selectivity. An analysis of the energetics of the -helix-channel-helix transition showed that the voltage-independent part of the free energy per helix residue could well be close to 0 kcal/mol and thus be in the range where a transition could occur. Two voltage-dependent contributions were included: the break down of the considerable dipole of the -helix and the outward shift of the positive charges of the side chains upon channel-helix formation. Taking into account the fact that the formation of an -helix is a highly cooperative process the degree of voltage dependence of the probability of formation of a channel-helix proved to be in the same range as experimental values for the open probability of modified Na channels whose inactivation had been removed. With regard to gating currents, the model predicts that 2.74 positive charges are moved in an outward direction. Consequences of the model for other experimental findings are discussed.     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.     

Stimulation of proliferation and immunoglobulin production of human-human hybridoma by various types of caseins and their protease digests

We have studied the effect of such milk proteins as caseins, lactalbumin, and lactoglobulin, on proliferation and immunoglobulin production of human-human hybridoma HB4C5 cells. It was found that -, -, and -caseins stimulated both proliferation and IgM product ion of human-human hybridoma HB4C5 cells, while the activities of -lactalbumin and -lactoglobulin were negligible. To localize the active sites of these caseins, effect of protease treatments on the activities were examined. When caseins were digested with trypsin, casein digests stimulated proliferation of the hybridoma, but not their IgM production. When -casein was digested with chymosin and fractionated top--casein and glycomacropeptide, both fragments stimulated proliferation of the cells, but onlyp--casein fragment stimulated IgM production. These results indicate that -casein has at least two proliferation stimulating sites and an IgM production stimulating site in thep--casein region.     

Water column and sediment nitrogen and phosphorus distribution patterns in the Florida Keys,USA

Measurements of the distribution patterns of nutrients (ammonium, nitrate, orthophosphate, total N and total P) and chlorophyll concentrations were conducted under an interdisciplinary program known as SEAKEYS, initiated because of concern that anthropogenic nutrients may be impacting Florida coral reefs. Samples were collected along transects that extended from passes or canals to 0.5 km offshore of the outermost reefs. Seven of the transects were either in the Biscayne National Park (BNP) and Key Largo (upper keys) or Seven Mile Bridge/Looe Key (upper part of lower keys) areas, which have the best present-day reef development; the two in the middle keys off Long Key were in an area of minimal reef development where passes allow estuarine Florida Bay water to flow onto the Florida reef platform. Off the upper keys, water column concentrations of N and chl a were elevated near marinas and canals (1 M NO₃, 1 g/l chl a), but returned to oligotrophic levels (e.g., chl a 0.25 g/l; NO₃ 0.25 M; NH₄ 0.10 M) within 0.5 km of shore. Phosphorus concentrations, however, were often higher offshore 0.2 M PO₄). Sediment interstitial nutrient concentrations decreased from inshore to the offshore reef areas (e.g., 100 M NH₄ inshore to 50 M NH₄ offshore) and were comparable to those of some presumably pristine coastal and reef carbonate sediments. Sediment bulk N was higher nearshore and decreased steeply offshore ( 60 g-at N/gm sediment to 20 g-at N/gm sediment, respectively); bulk P concentrations ( 6 g- at P/gm sediment) varied little or exhibited the reverse pattern. Sediment N:P ratios were consistently lower offshore (1–10 vs. 20–40 nearshore). Higher offshore P concentrations are attributed to periodic upwelling along the shelf edge. In the middle keys water column nutrients and chl a concentrations were both higher than those in the upper keys, and there was less of an inshore-offshore decrease than that noted in the upper keys. Sediment nutrients were higher also, and nearshore and offshore areas did not differ. Water column and sediment nutrient concentrations and distribution patterns in the upper part of the lower keys were most similar to those measured in the upper keys. Overall, the present data do not support the contention that reef areas in the upper keys are accumulating elevated loads of land-derived nutrients via surface water flow, but does document moderately elevated nutrient and chl a levels in many developed nearshore areas. Most of the anthropogenic and natural nutrients entering the coastal waters from shore appear to be taken up by near shore algal and seagrass communities before they reach patch reef areas. Further work is needed to determine whether nutrient-enriched ground waters reach the reefs, however these would be expected to cause an enrichment of reef sediments, which was not observed.     

Aflatoxin influences on seed germination and root elongation by two cultivars of Glycine max and uptake of 65 Zn-ZnCl2 by the cultivars

Maximum inhibition of Glycine max, cv. Essex seed germination occurred at 10 g/ml following 72 hr imbibition in constant light. Seeds imbided 108 hr in constant darkness at this concentration showed a 20% rise in germination over that of the control. Imbibition of G. max, cv. Williams seeds in either light or dark for 96 hr did not suppress germination. Imbibition of Essex seeds in either light or dark at 2.5 through 10 g/ml stimulated root elongation except for 10 g/ml at 96 hr (light). Maximum inhibition of Williams root elongation under constant light was at 48 and 72 hr with 10 g/ml. Statistically significant differences in cotyledon, leaf and stem lengths between non-treated (NT) and treated (T) seedlings were not found except for Williams stem length at 2.5 / ml. Root elongation was stimulated 1.2- and 1.1-folds, respectively, at 5.0 (Essex) and 2.5 (Williams) g/ml. Toxin at 2.5 through 10.0 g/ml did not markedly alter either cotyledon or leaf widths with the exception of Williams leaf width at 2.5 g/ml. Medium supplementation with 2.5 through 10.0 g/ml resulted in cotyledon, leaf and root weight enhancements for Essex seedlings. Stem weight was not markedly affected. An 18% rise in Williams cotyledon weight above that of the control was seen at 2.5 g/ml. Williams leaf weights were increased 1.75- and 1.25-folds, respectively, at 2.5 and 10.0 g/ml. Aflatoxin B₁, at 2.5 g/ml promoted Williams stem and root elongation 1.20- and 1.09-folds, respectively. Most of the radioactivity from ⁶⁵Zn-ZnCl₂ recovered within organs was found within Essex roots for both T and NT seedlings. A higher amount of radioactivity was recovered within roots at each toxin concentration than was without toxin. However, this was not statistically significant. Significant differences in the distribution of radioactivity within roots between NT and T Williams seedlings were not observed. Generally, AFB₁ failed to affect significantly these two varieties of soybeans based on the tests relating to germination, growth and radiolabel uptake.