A carbonic anhydrase inhibitor induces bicarbonate-reversible suppression of electron transfer in pea photosystem 2 membrane fragments.

The effects of suppression of the carbonic anhydrase (CA) activity by a CA-inhibitor, acetazolamide (AA), on the photosynthetic activities of photosystem II (PS II) particles from higher plants were investigated. AA along with CA-activity inhibits the PS II photosynthetic electron transfer and the AA-induced suppression is totally reversed by the addition of bicarbonate (3-5 mM). Similar effect of recovery in the PS II photosynthetic activity was also revealed upon the addition of known artificial electron donors (potassium ferrocyanide and TMPD). Significance and possible functions of CA for the PS II donor side are discussed.

Incidence and risk factors for depression and anxiety disorders: results from a 34-year longitudinal Swedish cohort study.

This study is based on a total cohort (N = 192) of people born in 1902 and 1903 and living in southern Sweden. Subjects were assessed at baseline when 67 years of age and on eight further occasions over 34 years or until death. The participation rate in the nine examinations ranged from 78-100%. Interviews, psychological tests, and medical examinations were used as well as information on medical diagnoses from primary health care records and hospital records. The cumulative probability for the development of clinical depression during the follow-up was 8% and for anxiety 6%. The incidence rate for depression and for anxiety was highest during the period 67-81 years. Persons with poor financial status were more likely to be diagnosed with depression but no significant risk factor for anxiety was found. Only 14% developed depression and anxiety during the follow-up period, females more often than men. The strongest risk factors for the development of depression were perceived economic problems.

Carbonic anhydrase activities in pea thylakoids.

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (mg Chl)(-1) h(-1) at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO(3) (-) dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 micromol H(+) (mg Chl)(-1) h(-1)) were observed in the presence of detergents (Triton X-100 or n-octyl-beta-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 micromol H(+) (mg Chl)(-1) h(-1) which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.

Formal support, mental disorders and personal characteristics: a 25-year follow-up study of a total cohort of older people.

The present study was designed to describe the pattern of long-term formal support received by people with mental disorders, and to investigate the relationship between the medical, psychological and social characteristics of the participants and types of formal support. This study is based on a cohort (n = 192) of people born in 1902 and 1903 in a community in Southern Sweden. The research participants were assessed using interviews, psychological tests and medical examinations. Information was collected about the use of primary healthcare and social services. The first assessment took place when the cohort was aged 67 years, and then on eight further occasions until they were 92. The participation rate ranged from 72% to 100%. During the observation period of 25 years, 53% of people with dementia eventually received both home help and institutional care compared to 34% of people with other psychiatric diagnoses and 12% of people with good mental health. The last group had all physical health problems and/or problems with activities of daily living. However, 35% of the dementia group, 46% with other psychiatric diagnoses and 52% of people with good mental health did not receive any formal support. Males and self-employed people were significantly less likely to use formal support. The institutionalised group reported loneliness significantly more often than the other two groups. In a logistic regression analysis, loneliness, low social class, high blood pressure and low problem-solving ability were predictors of formal support use. People with a mental disorder, including dementia, were significantly more likely to use formal support compared to people with good mental health. Social factors were the main factors predicting formal support.

The gender differences in growth hormone-binding protein and leptin persist in 80-year-old men and women and is not caused by sex hormones.

OBJECTIVE: Leptin and growth hormone-binding protein (GHBP) both show gender differences that might be explained by sex hormones. To study the potential relevance of oestradiol and testosterone, we have examined 80-year-old subjects in whom oestradiol is higher in men than in women. The interrelationships between leptin, insulin, GHBP and fat mass in this age group were also investigated. DESIGN AND SUBJECTS: Ninety-four subjects (55 females and 39 males), all 80 years old, were investigated in a community-based study. None of the investigated subjects was being treated for diabetes mellitus and none of the women had oestrogen replacement. METHODS: Levels of testosterone, oestradiol, SHBG, IGF-I, GHBP, glucose, insulin and leptin were analysed. Body composition was measured with bioimpedance analysis (BIA). RESULTS: As in younger age groups, serum leptin, the ratio leptin/kilogram fat mass and serum GHBP were higher in the women (all, P< or =0.007), although serum oestradiol was higher in the men (P<0.001). There were no significant associations between sex hormones and leptin or GHBP either in women or in men (all, r<0.13, P>0.1). Leptin correlated to kilogram fat mass in both women (r=0.55, P<0.001) and men (r=0.47, P=0.003), but in contrast, there were no significant correlations between GHBP and fat mass and GHBP and IGF-I, either in women or in men (all, r<0.24, P>0.2). Insulin and leptin were significantly associated with GHBP, both in women (r=0.48, P<0.001 and r=0.43, P=0.001, respectively) and in men (r=0.40, P=0.01 and r=0.34, P=0.03, respectively). CONCLUSIONS: Although the 80-year-old men had higher oestradiol levels than the women, the women had higher levels of leptin and GHBP. There were no correlations between sex hormones and leptin and GHBP, which indicates that the gender differences are not caused by sex hormones in old age. In contrast to studies in younger subjects, GHBP did not correlate to fat mass in the investigated 80-year-old men and women. In the older subjects investigated, as in younger subjects, GHBP was significantly correlated with leptin and insulin.

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus.

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp(241) is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310-395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.

Small, novel proteins from the mistletoe Phoradendron tomentosum exhibit highly selective cytotoxicity to human breast cancer cells.

Four novel proteins (phoratoxins C-F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC50 values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples.

Client expectations and satisfaction of quality in home care services. A consumer perspective.

This study examines clients' expectations of quality in home care services and their perceived satisfaction with services among a random sample of 76 home care recipients in Vancouver, Canada. The researchers conducted face-to-face interviews that applied Multiattribute Utility Technology, a procedure that organizes several quality attributes of "ideal" home care into a tree structure to compare their relative importance and ranking from the clients' perspective. Participants also were asked to state their satisfaction or dissatisfaction with the services received in these domains. Among the five main quality attributes identified, the subjects ranked suitability of the home helper and its subset, personal competence, as the most important indicators of quality, followed by continuity in service. In addition, clients tended to have a high level of satisfaction with regard to the attributes of overall home care services. The highest level of satisfaction was reported for elements of personal dispositions of home care staff. The lowest level of satisfaction involved the time/availability components of the service. Finally, comparisons between client expectations and satisfaction of received home care services showed the highest discrepancy for the attributes of influence and time/availability and the greatest congruence for personal attributes of the staff. The results are discussed in terms of their implications for the delivery of home care services.

Social relationships and health as predictors of life satisfaction in advanced old age: results from a Swedish longitudinal study.

This longitudinal study examines the relationship between family and friend social support, health, and life satisfaction for a single cohort of eighty-year-old persons living in Lund, Sweden. Results indicate that participants who remained in the study are healthier and score higher on life satisfaction when compared with those who either drop-out or die prior to age eighty-three. Even though well-integrated with family and friends, the number of friends decreases significantly from eighty to eighty-three years; those who reported no close friends nearly doubled from eighty to eighty-three years. However, for those with close friends, contact with friends increases with age. In contrast to previous research, a correlational analysis indicates that neither child nor friend support is related to life satisfaction at either eighty or eighty-three years. However, health measures and satisfaction with sibling contact are related to total life satisfaction at age eighty-three only. These findings indicate the multidimensionality of both social support and life satisfaction for the old-old.

Role of a novel photosystem II-associated carbonic anhydrase in photosynthetic carbon assimilation in Chlamydomonas reinhardtii.

Intracellular carbonic anhydrases (CA) in aquatic photosynthetic organisms are involved in the CO2-concentrating mechanism (CCM), which helps to overcome CO2 limitation in the environment. In the green alga Chlamydomonas reinhardtii, this CCM is initiated and maintained by the pH gradient created across the chloroplast thylakoid membranes by photosystem (PS) II-mediated electron transport. We show here that photosynthesis is stimulated by a novel, intracellular alpha-CA bound to the chloroplast thylakoids. It is associated with PSII on the lumenal side of the thylakoid membranes. We demonstrate that PSII in association with this lumenal CA operates to provide an ample flux of CO2 for carboxylation.

Concentrations of plasma methylmalonic acid in 80-year-olds show only weak relation to psychological performance.

Most studies of the relation between psychological performance in the elderly and deficiencies of cobalamin and folate have used methods that determine the blood concentrations of these vitamins, which might not reflect the vitamin status in the tissues. Recently, two new markers, plasma homocysteine and methylmalonic acid, have attracted growing interest since they are considered to reflect the status of cobalamins and folates in the tissues. In a previous study, we noted a strong association between five parameters of well-being and lower concentrations of plasma homocysteine. In the present study, we have extended these observations by determination of plasma methylmalonic acid in the same healthy elderly population. In the present study, 18 out of 100 subjects had increased plasma methylmalonic acid and in 7 of these subjects, the concentrations of serum cobalamin, blood folate, plasma homocysteine and serum creatinine were within normal limits. The relation between plasma methylmalonic acid concentrations and concentrations of serum cobalamin and blood folates and five parameters of well-being were investigated. Concentrations of plasma methylmalonic acid were only weakly associated with the concentrations of serum cobalamin and lower scores on the logical reasoning test. The present study clearly shows that the levels of plasma methylmalonic acid show a much lesser association with the parameters of well-being than did plasma homocysteine.

Insomnia in an 80-year-old population: relationship to medical, psychological and social factors.

In this study, insomnia in 80-year-olds was related to medical, psychological and social factors. The data were based on examinations every year in people aged between 80 and 89 years. Of 333 people living in the city of Lund and born in 1908, 67% participated. Increased severity of insomnia was significantly associated with use of diuretics, other cardiovascular drugs, hypnotics and laxatives, and with nervousness, difficulty relaxing, anorexia, nausea, constipation, backache, feeling cold, sweating, loss of weight, dizziness, depression, general fatigue, exhaustion, angina pectoris, cardiac insufficiency, worsened objective and subjective health, presence of negative T-waves on ECG, anxiety, total life satisfaction, neuroticism, disbelief in a just world, feeling lonely and lower survival rates. Thus insomnia has widespread associations with different aspects of life in 80-year-olds.

Intracellular beta-carbonic anhydrase of the unicellular green alga Coccomyxa. Cloning of the cdna and characterization of the functional enzyme overexpressed in Escherichia coli.

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular beta-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to beta-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.

A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.

Induction and Regulation of Expression of a Low-CO2-Induced Mitochondrial Carbonic Anhydrase in Chlamydomonas reinhardtii

The time course of and the influence of light intensity and light quality on the induction of a mitochondrial carbonic anhydrase (CA) in the unicellular green alga Chlamydomonas reinhardtii was characterized using western and northern blots. This CA was expressed only under low-CO2 conditions (ambient air). In asynchronously grown cells, the mRNA was detected 15 min after transfer from air containing 5% CO2 to ambient air, and the 21-kD polypeptide was detected on western blots after 1 h. When transferred back to air containing 5% CO2, the mRNA disappeared within 1 h and the polypeptide was degraded within 3 d. Photosynthesis was required for the induction in asynchronous cultures. The induction increased with light up to 500 mumol m-2 s-1, where saturation occurred. In cells grown synchronously, however, expression of the mitochondrial CA was also detected in darkness. Under such conditions the expression followed a circadian rhythm, with mRNA appearing in the dark 30 min before the light was turned on. Algae left in darkness continued this rhythm for several days.

Plasma homocysteine in 80-year-olds: relationships to medical, psychological and social variables.

Plasma homocysteine concentrations in a group of 80-year-old persons were related to symptoms and signs. Plasma homocysteine concentrations higher than 15 micromol/l were associated with lower total life satisfaction (P<0.01), mood (P<0.05), zest for life (P<0.05), lower scores for reasoning (P<0.05), spatial ability (P<0.05), memory recognition (P<0.05), and subjective health (P<0.01). In an instrument comprising of 30 symptoms, plasma homocysteine concentrations higher than 15 micromol/l were associated with impaired concentration (P<0.05), restlessness (P<0.05), feeling cold (P<0.05), loss of weight (P<0.05), and feeling depressed (P<0.01). The above data indicate that plasma homocysteine values over 15 micromol/l could be relevant markers for clinical intervention.

Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii.

Nuclear genes coding for carbonic anhydrase, a major mitochondrial constituent in Chlamydomonas reinhardtii grown under limited CO2, were characterized. Two genes, ca1 and ca2, were found within 7 kb of genomic DNA, organized 'head to head' in a large inverted repeat. The DNA sequences for the two genes were very similar, even in the promoter regions and in introns, indicating that the repeat is a result of a recent duplication. To study gene regulation, elements from the upstream region of ca1 were fused to the arylsulphatase reporter gene. After transformation, the expression of arylsulphatase was regulated similarly to the endogenous ca1/ca2 genes, even when the promoter was trimmed down to 194 nt. Expression could not be detected when 5% CO2 was bubbled into the growth medium, but was induced within hours after transfer to air. The ca1 promoter was not induced in low light, but at intermediate light levels its activity was dependent on the irradiance. O2 concentration had no effect on the promoter activity, indicating that photorespiratory metabolites are not triggering the response. The availability of cells transformed with a CO2-regulated reporter gene should facilitate further studies on the metabolic adaptations that occur in some green algae in response to the external CO2 level.