Characterization of the near-surface layers of carbon steels modified by interaction with intense pulsed plasma beams: scanning electron microscopy investigations.

Modification of materials is a wide area in materials science, especially surface modification. To investigate the results of the modification process, treated and nontreated samples were compared. Intense plasma pulses of argon or nitrogen were used to irradiate the carbon steels. In all samples, the near-surface layer was melted. Results of scanning electron microscopy investigations of the surface morphology and cross-sections, as well as the results of tribological tests, are presented. The obtained results allowed us to draw conclusions about changes in material properties and to propose subsequent studies using other investigation techniques.

Iodine deficiency disorders incidence in neonates based on the experience with mass screening for congenital hypothyroidism in southeast Poland in the years 1985-2000.

Thyroid hormones are very important for the child mental and physical development. In southeast Poland screening for congenital hypothyroidism detection was established in 1985; this region was defined as an iodine deficient area. The results provided the first signal pointing to insufficient iodine prophylaxis in the region. In the years 1985-2000, a total of 634,179 newborns were tested. TSH levels in blood spots on filter paper were determined using three consecutive methods: RIA, fluoroimmunometry (FIA) and luminoimmunemetry (LIA). In the first five years of the program, the incidence of permanent hypothyroidism was 1:4,000, while the incidence of iodine deficiency disorders (IDD) was 1:272. These findings led to the recognition of the fact that iodine prophylaxis had been discontinued in 1981 and reintroduced in 1992 and from that time on the incidence of IDD in neonates has been evidently lower. In the period between 1991 and 2000, we observed a 0.14% incidence rate of IDD, while until 1991 it had been 0.5%. In the years 1998-2000, the respective value was 1:5,420. The incidence of transient hypothyroidism also dropped from 1:3,920 to 1:48,474. Therefore we conclude that, based on screening studies carried out since 1985, the analysis of TSH levels may be regarded as a tool for iodine deficiency monitoring in neonates. Between 1985-2000, a drop in the incidence of IDD in newborns is clearly seen. Moreover, investigations carried out in particular years show that even low-grade iodine supplementation leads to TSH level decrease in newborns.

Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.

The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L.

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition.

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H(+)-transport by H(+)-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, Deltamu(H+), across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J(mal)(rel), of apparent ATP-dependent malate-stimulated H(+)-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J(mal)(rel) was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO(3)(-) nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J(mal)(rel). It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO(3)(-) nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins.

Processing of V-ATPase subunit B of Mesembryanthemum crystallinum L. is mediated in vitro by a protease and/or reactive oxygen species.

Soluble proteins were isolated from leaves of the common ice plant Mesembryanthemum crystallinum L. in the CAM state of photosynthesis and tested for protease activity using amino acid-beta-naphthylamide (NA)-derivatives in a search for proteolytic activity responsible for cleavage of the V-ATPase subunit B. This cleavage is suggested to occur at the peptide bond between Met192 and Glu193. At neutral pH Met-NA was one of seven derivatives which were cleaved by proteases present in this fraction. Enzymes exhibiting proteolytic activity were separated from other soluble proteins by Superose 12-size exclusion FPLC. Incubation of partially purified protease with tonoplast-enriched membrane vesicle fractions isolated from M. crystallinum in the C3-state of photosynthesis led to a decrease in subunit B (55 kDa) protein amount and to the formation of the polypeptide Di (32 kDa), which has been previously suggested to represent a fragment of subunit B. Cleavage of subunit B and the appearance of Di also occurred during incubation of tonoplast vesicles in the presence of reactive oxygen species. In addition to Di, the polypeptide Ei (28 kDa) appeared after incubation with protease and/or reactive oxygen species. Taken into account that Di and Ei cross-reacted with an affinity purified antiserum directed against subunit B, Di as well as Ei might represent fragments of subunit B. These results open new perspectives with respect to the regulation of V-ATPase modification and turnover.

Structure, function and regulation of the plant vacuolar H(+)-translocating ATPase.

The plant V-ATPase is a primary-active proton pump present at various components of the endomembrane system. It is assembled by different protein subunits which are located in two major domains, the membrane-integral V(o)-domain and the membrane peripheral V(1)-domain. At the plant vacuole the V-ATPase is responsible for energization of transport of ions and metabolites, and thus the V-ATPase is important as a 'house-keeping' and as a stress response enzyme. It has been shown that transcript and protein amount of the V-ATPase are regulated depending on metabolic conditions indicating that the expression of V-ATPase subunit is highly regulated. Moreover, there is increasing evidence that modulation of the holoenzyme structure might influence V-ATPase activity.

The role of selenium in iodine metabolism in children with goiter.

Possible interactions between selenium and iodine metabolism were investigated in 7- to 16-year-old children with goiter (n = 136) living in southeastern Poland in iodine-deficient areas influenced by a sulfur industry. The Se-iodine interactions in these children were compared to the interactions in children from outside of that region (n = 38). Blood selenium (BSe) concentration and plasma glutathione peroxidase activity were much lower in the study group (64.1 +/- 15.7 microg/L; 111.0 +/- 27.6 U/L) than in the control group (85.3 +/- 19.6 microg/L; 182.4 +/- 35.6 U/L). Almost all of the data [plasma thyroid-stimulating hormone (TSH) concentration, plasma free thyroxine (fT(4)) concentration] fell within the reference limits. There was no statistically significant difference between the control and the study groups with respect to fT(4) and TSH. However, statistically significant differences of fT(4) and TSH in the study group were revealed between females belonging to the lower (n = 21; fT(4), 16.1 +/- 3.3 pmol/L; TSH, 1.83 +/- 1.05 mU/L) and upper Se quartiles (n = 24; fT(4), 14.5 +/- 2.2 pmol/L; TSH, 1. 26 +/- 0.90 mU/L), p < 0.05. Neither group differed in iodine in urine concentration, age, and body mass index. The difference in fT(4) concentrations can be attributed to an Se deficiency. The relationship exists only for females, which suggests a sex-linked hormonal response to concomitant Se and iodine deficiencies.

Localization of pyrophosphatase in membranes of cauliflower inflorescence cells.

Using a polyclonal antiserum specific for the tonoplastic H(+)-pyrophosphatase (tPPase), significant amounts of antigenic polypeptides of the correct molecular mass were detected in Western blots of plasma membrane isolated from cauliflower (Brassica oleracea L.) inflorescence by phase-partitioning and subsequent sucrose density centrifugation. Potassium iodide-stripped plasma membranes continued to give a strong positive signal, indicating that the PPase antigen detected was not a result of contamination through soluble PPase released during homogenisation. The same preparation contained negligible vacuolar (v)H(+)-ATPase activity and the A subunit of the vATPase could not be detected by immunoblotting. Plasma membrane fractions exhibited a proton-pumping activity with ATP as substrate, but such an activity was not measurable with pyrophosphate, although the hydrolysis of this substrate was recorded. By contrast, pyrophosphate supported proton pumping in tonoplast-containing fractions. Immunogold electron microscopy confirmed the presence of PPase at the plasma membrane as well as at the tonoplast, trans Golgi network, and multivesicular bodies. The density of immunogold label was higher at the plasma membrane than at the tonoplast, except for membrane fragments occurring in the lumen of the vacuoles which stained very conspicuously.

Catalase activity during C3-CAM transition in Mesembryanthemum crystallinum L. leaves.

Treatment with 0.4 mol dm(-3) NaCl caused a C3-CAM shift in Mesembryanthemum crystallinum L. leaves. In parallel to the CAM induction the activity of CAT was significantly decreased. In C3 and in CAM plants CAT activity showed daily fluctuations, with the maximum at the end of the light period. The oscillations of CAT were more pronounced in CAM than in C3 plants. In M. crystallinum CAT activity seems to respond more to CAM induction than to salinity.

Mass screening program for congenital hypothyroidism in south-eastern Poland.

The success of a screening program depends in great measure on effective organization. The purpose of the screening program is the commencement of treatment of selected patients within two weeks of life. In total in our laboratory we have tested 461,479 newborns during 1985-1996. The introduction of bar codes in 1995 (recommended by the Institute of Mother and Child in Warsaw) improved our work in the laboratory and departments of obstetrics and reduced the possibility of errors. The screening tests for congenital hypothyroidism appear to serve the monitoring of iodine deficiency disorders. The good organization of the screening program is indispensable and needs permanent control.

Phenotypic adaptation of tonoplast fluidity to growth temperature in the CAM plant Kalanchoë daigremontiana ham. et Per. is accompanied by changes in the membrane phospholipid and protein composition.

The present study deals with the phenotypic adaptation of tonoplast fluidity in the CAM plant Kalanchoë daigremontiana to changes in growth temperature. Tonoplast fluidity was characterized by measuring fluorescence depolarization in membranes labeled with fluorescent fatty acid analogues and by following formation of eximeres in membranes labeled by eximere-forming fluorophores. With both techniques it was found that exposure of the plants to higher growth temperature compared with the control decreased the fluidity of the tonoplast while exposure to lower growth temperature caused the opposite. Three hours of high temperature treatment (raised from 25 degreesC to 35 degreesC; "heat shock") were sufficient to decrease the tonoplast fluidity to roughly the same extent as growth under high temperature for 30 days. The phenotypic response of tonoplast fluidity to changes in growth temperature was found only in the complete membrane, not however in the lipid matrix deprived of the membrane proteins. Heat treatments of the plants decreased the lipid/protein ratio while exposure to low temperature (for 30 days) increased it. Heat treatments led to a decrease in the percentage of linolenic acid (C18:3) and linoleic acid (C18:2), heat shock and low temperature treatments induced an increase in the percentage of linoleic acid (C18:3), with concomitant decrease in the percentage of linoleic acid (C18:2). However, in the case of heat shock, increase in linolenic acid concerned mainly monogalactosyldiacylglycerol, while with low temperature treatment linoleic acid increased in phosphatidylcholine. Both treatment of the plants with high and low temperature led to a slight decrease in the contribution of phosphatidylcholine and phosphoethanolamine to the total phospholipid content of the tonoplast. High-temperature treatment of the plants not only decreased the phospholipid/protein ratio in the tonoplast, but also led to the occurrence of a 35 kDa polypeptide in the tonoplast which cross-reacted with an antiserum against the tonoplast H+-ATPase holoenzyme. The important role of membrane proteins in bringing about the phenotypic rigidization of the tonoplast was mimicked by reconstitution experiments showing that incorporation of the proteins isolated from the tonoplast into phosphatidylcholine vesicles decreased the fluidity of this membrane system. As to be expected from the analyses in the natural membrane, the degree of this effect depended on the phospholipid/protein ratio.

Increased Expression of Vacuolar Aquaporin and H+-ATPase Related to Motor Cell Function in Mimosa pudica L.

Mature motor cells of Mimosa pudica that exhibit large and rapid turgor variations in response to external stimuli are characterized by two distinct types of vacuoles, one containing large amounts of tannins (tannin vacuole) and one without tannins (colloidal or aqueous vacuole). In these highly specialized cells we measured the abundance of two tonoplast proteins, a putative water-channel protein (aquaporin belonging to the [gamma]-TIPs [tonoplast intrinsic proteins]) and the catalytic A-subunit of H+-ATPase, using either high-pressure freezing or chemical fixation and immunolocalization. [gamma]-TIP aquaporin was detected almost exclusively in the tonoplast of the colloidal vacuole, and the H+-ATPase was also mainly localized in the membrane of the same vacuole. Cortex cells of young pulvini cannot change shape rapidly. Development of the pulvinus into a motor organ was accompanied by a more than 3-fold increase per length unit of membrane in the abundance of both aquaporin and H+-ATPase cross-reacting protein. These results indicate that facilitated water fluxes across the vacuolar membrane and energization of the vacuole play a central role in these motor cells.

Differential immunological cross-reactions with antisera against the V-ATPase of Kalanchoë daigremontiana reveal structural differences of V-ATPase subunits of different plant species.

Two antisera (ATP88 and ATP95) raised against the V-ATPase holoenzyme of Kalanchoë daigremontiana were tested for their cross-reactivity with subunits of V-ATPases from other plant species. V-ATPases from Kalanchoë blossfeldiana, Mesembryanthemum crystallinum, Nicotiana tabacum, Lycopersicon esculentum, Citrus limon, Lemna gibba, Hordeum vulgare and Zea mays were immunoprecipitated with an antiserum against the catalytic V-ATPase subunit A of M. crystallinum. As shown by silver staining and Western blot analysis with ATP88, subunits A, B, C, D and c were present in all immunoprecipitated V-ATPases. In contrast, ATP95 recognized the whole set of subunits only in K. blossfeldiana, M. crystallinum, H. vulgare and Z. mays. This differential cross reactivity of ATP95 indicates the presence of structural differences of certain V-ATPase subunits. Based on the Bafilomycin A1-sensitive ATPase activity of tonoplast enriched vesicles, and on the amount of V-ATPase solubilized and immunoprecipitated, the specific ATP-hydrolysis activity of the V-ATPases under test was determined. The structural differences correlate with the ability of V-ATPases from different species to hydrolyze ATP at one given assay condition for ATP-hydrolysis measurements. Interestingly V-ATPases showing cross-reactivity of subunits A, B, C, D and c with ATP95 showed higher rates of specific ATP hydrolysis compared to V-ATPases containing subunits which were not labeled by ATP95. Thus, V-ATPases with high turnover rates in our assay conditions may show common structural characteristics which separate them from ATPases with low turnover rates.

The 32 kDa tonoplast polypeptide Di associated with the V-type H+-ATPase of Mesembryanthemum crystallinum L. in the CAM state: A proteolytically processed subunit B?

In the facultative halophyte Mesembryanthemum crystallinum, the salt- or age-induced transition to crassulacean acid metabolism (CAM) leads to the occurrence of a tonoplast-bound 32 kDa polypeptide (Di). The alignment of its N-terminal protein sequence with protein sequences of recently cloned higher plant V-ATPase B-subunits indicates that Di may be derived from subunit B by proteolytic removal of a protein fragment of about 20 kDa from its N-terminus. Furthermore, an antiserum directed against Di cross-reacts with subunit B from Nicotiana tabacum. It inhibits both proton pumping and ATP hydrolysis of the holoenzyme in M. crystallinum. As Di remains firmly attached to the holoenzyme the proteolytic processing may have functional implications.

Early salt stress effects on the differential expression of vacuolar H(+)-ATPase genes in roots and leaves of Mesembryanthemum crystallinum.

In Mesembryanthemum crystallinum, the salt stress-induced metabolic switch from C3 photosynthesis to Crassulacean acid metabolism is accompanied by major changes in gene expression. However, early effects of salt exposure (i.e. prior to Crassulacean acid metabolism induction) on genes coding for vacuolar transport functions have not yet been studied. Therefore, the expression of vacuolar H(+)-ATPase genes was analyzed in different organs of 4-week-old plants stressed with 400 mM NaCl for 3, 8, or 24 h. Partial cDNAs for the subunits A, B, and c were cloned and used as homologous probes for northern blot analysis. In control plants, the mRNA levels for the different subunits showed organ-specific differences. In fully expanded leaves, subunit c mRNA was very low but increased transiently during the light period. Plant organs also differed in their salt-stress response. In roots and young leaves, mRNA levels for all three subunits increased about 2-fold compared to control plants, whereas in fully expanded leaves only subunit c mRNA responded to salt. The results indicate that the expression of vacuolar H(+)-ATPase genes does not always involve a fixed stoichiometry of mRNAs for the different subunits and that the mRNA level for subunit c is particularly sensitive to developmental and environmental changes.

Thyroid dysfunction in newborns and infants living in an iodine-deficient area.

The region of Southern Poland is known as an iodine-deficient area. Iodine prophylaxis was discontinued in 1980/81. This fact was discovered as a result of the introduction of newborn mass screening for congenital hypothyroidism in 1985. In view of the large number of newly detected cases of Iodine Deficiency Disorders (IDD), we compared the neonatal populations of the Cracow and Nowy Sacz provinces. The prevalence of IDD was revealed as eight times higher in Nowy Sacz than in Cracow. The iodine urine excretion rates in those two provinces were similar. The paper discusses the cause of the discrepancy in IDD incidence. The final results show that the iodine deficiency detected in the Cracow and Nowy Sacz provinces is of moderate degree. Salt iodination in the region of southern Poland needs a new approach even before the year 2000, as planned in the WHO program.

Goiter incidence and urinary iodine excretion in children of age group 6-13 years living in south-eastern Poland (Kraków coordinating center).

The study included 4103 school children attending randomly selected schools localized in towns and villages of 3 geographically distinct regions: mountainous, (Carpathian endemia), upland, lowland. The children were divided into four age-related groups. The incidence of goiter as detected by palpation was 38.1% in all the children studied, the value being similar in all three regions (38.1, 37.3 and 38.6%, respectively). Goiter incidence differs slightly among urban and rural children, amounting to 40.5 and 34.5%, respectively, in the two groups. In about 60% of children urinary iodine excretion was lower than 50 micrograms/l, while in about 30% it was normal. In about 10% of children studied urinary iodine excretion was lower than 20 micrograms/l. The results obtained indicate the occurrence among children of the studied region of goiter endemy of moderate degree. It was found that the percentage of children excreting more than 50 micrograms/l of urinary iodine was higher among those consuming iodized salt as compared to those consuming non-iodized salt. The first effects of resumption of common salt fortification with iodide in southern Poland can already be noted. There is, however, a need for optimization and steady control over this prophylactic action.

[Dietary iodine intake in children and clinical and biochemical features of iodine deficiency in chosen districts of south-east Poland]. / Spozycie jodu w pokarmach dzieci a kliniczne I biochemiczne cechy niedoboru jodu w wybranych województwach Polski Poludniowo-wschodniej.

The study on the effects of Czarnobyl accident on thyroid gland function in children (MZ XVII program) revealed a high incidence of goiter in the population of children in district Kraków and Nowy Sacz. Other study on Tarnobrzeg population showed that the frequency of goiter in school children was 67%. The goiter prevalence, urine iodine excretion and iodine food consumption in the same populations were compared. The detailed investigation of iodine intake in children by feeding questionnaire shows a low consumption of iodine. The urine iodine excretion in the population of Kraków-district was low, but higher than in Nowy Sacz-district and nearly normal in Tarnobrzeg. High frequency of goiter in children and of IDD in newborns screened for CH, as well as low urine iodine excretion, together with low iodine intake with food are the markers of iodine deficiency in Kraków and Nowy Sacz districts. The improvement of feeding as well as iodine salt supplementation is necessary. The situation in Tarnobrzeg district looks differently and needs additional study.

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids.

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.