Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria.

Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.

Mid-term Outcomes of the Supported Ross Procedure in Children, Teenagers, and Young Adults.

The Ross procedure in children, teenagers, and young adults, especially among those with a bicuspid aortic valve with aortic regurgitation, has been associated with neoaortic root dilatation and recurrence of aortic regurgitation. We have shown that at intermediate follow-up, patients who underwent a supported Ross technique were less likely to have neoaortic root dilatation. This study summarizes our most recent outcomes. This was a retrospective review of 40 consecutive pediatric and young adult patients undergoing a supported Ross procedure from 2005 to 2018. Clinical outcomes were reviewed in addition to echocardiographic measures of neoaortic dimension and neoaortic valve function. The median age at surgery was 16.0 years (range 10 - 35 years). Preoperative diagnosis was aortic regurgitation in 15 (37.5%) and mixed regurgitation/stenosis in 20 (50%). Median follow-up was 3.5 years (1.4-5.6) with 3 patients followed for more than 10 years. There were no deaths. Five patients had a reintervention, but only 1 on the aortic valve. One patient returned to the operating room on postoperative day 1 for revision of the right coronary button. Two patients required biventricular pacemakers for reduced ejection fraction. One patient developed aortic regurgitation and underwent mechanical valve replacement and another required a reintervention on the homograft with a percutaneously placed pulmonary valve replacement. At last follow-up, 39 patients had mild or less aortic regurgitation with median sinus z-score of 1.40 (0.48-2.07). Mid-term follow-up of pediatric and young adult patients undergoing a supported Ross operation for various aortic valve pathologies demonstrate excellent results with minimal neoaortic root dilation and reintervention.

Effects of peripheral administration of synthetic human glucose-dependent insulinotropic peptide (GIP) on energy expenditure and subjective appetite sensations in healthy normal weight subjects and obese patients with type 2 diabetes.

BACKGROUND: Apart from their role in insulin secretion and glucose homeostasis, the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) exert a number of extra-pancreatic effects which in the case of GIP remain largely unknown. DESIGN/PATIENTS: Six obese male patients with diet-controlled type 2 diabetes (T2DM) and six healthy lean male subjects were studied. The protocol included four experiments for each participant that were carried out in randomized order and included: GLP-1 infusion at a rate of 1 pmol/kg/min for 4 h, GIP at a rate of 2 pmol/kg/min, GLP-1 (at 1 pmol/kg/min) with GIP (at 2 pmol/kg/min), and placebo infusion for 4 h. Energy expenditure (EE) was measured throughout with indirect calorimetry and subjects were given a series of visual analogue scales to rate hourly their hunger, fullness, urge to eat and prospective consumption of food. Immediately following termination of the infusions all subjects were offered a free choice buffet lunch and total calorie and macronutrient intake was calculated. RESULTS: During GIP infusion there was a trend for healthy subjects to report higher hunger scores and a reduction in EE only when compared with placebo. These parameters remained unchanged in patients with T2DM. Ad libitum energy intake after all four infusions was the same in both groups. CONCLUSION: We report here for the first time that GIP infusion may impact on resting EE and subjective appetite sensations in normal weight healthy subjects and further studies with larger numbers of subjects are required to help define more conclusively the precise role of GIP in energy balance in humans.

Plasma obestatin levels are lower in obese and post-gastrectomy subjects, but do not change in response to a meal.

OBJECTIVE: To investigate a potential role for obestatin in humans by examining response to a fixed energy meal. CONTEXT: A new anorectic peptide hormone, obestatin has recently been isolated from rat stomach. The significance of this peptide in humans is unknown. STUDY DESIGN: Case-control study. SETTING: Hospital-based study. PATIENTS: Nine healthy controls, nine morbidly obese subjects and eight post-gastrectomy subjects. INTERVENTION: Subjects attended after an overnight fast and were given a fixed energy meal (1550 kJ). MAIN OUTCOME MEASURE: The response of obestatin to a meal in the different groups. RESULTS: Fasting obestatin was significantly lower in obese subjects as compared to lean subjects (27.8+/-4 vs 17.2+/-2 pg/ml, P=0.03). Obestatin was also decreased in gastrectomy subjects but this did not reach statistical significance (27.8+/-4 vs 21.9+/-3 pg/ml, P=0.3). Obestatin did not change significantly from baseline in response to the meal. Lean and obese subjects had a similar obestatin/ghrelin ratio (0.04+/-0.003 vs 0.05+/-0.009, P=0.32), but this was higher in the gastrectomy group (0.04+/-0.003 vs 0.1+/-0.01, P<0.001). CONCLUSIONS: Obestatin does not vary significantly with a fixed energy meal, but is significantly lower in morbidly obese subjects as compared to lean subjects supporting a possible role for obestatin in long-term body weight regulation. Obestatin tended to be lower in gastrectomy subjects and their obestatin/ghrelin ratio differed from healthy controls. Hence, the expression of obestatin is altered following gastrectomy, suggesting other sites outside the stomach may also secrete obestatin.

Synthesis and Characterization of Ruthenium(II) Phenanthroline Complexes Containing Quaternary Amine Substituents.

A series of mixed ligand complexes of ruthenium(II) complexes containing 5-methylphenanthroline and trimethylamino-5-methylphenanthroline have been synthesized to investigate the impact of the quaternary amine on the photophysical properties. Thermal stability studies indicate that the quaternary amine group is stable with respect to hydrolysis. Mass spectral analysis of the complexes revealed only fragments consistent with homolytic cleavage of the amines and no parent ions were observed. Both electrochemical and photophysical investigations indicate that the quaternary amine has little or no impact on the properties of the complex when compared to the complexes lacking the amine.

The circadian rhythm of osteoprotegerin and its association with parathyroid hormone secretion.

BACKGROUND: Osteoclast resorptive activity, which is known to demonstrate circadian rhythmicity, is regulated by various endocrine hormones and cytokines. PTH suppresses osteoprotegerin (OPG), a regulator of osteoclast activity that has recently been shown to have a circadian rhythm in healthy controls. We studied the differences in the relationship between PTH, OPG, and type I collagen C-telopeptide (betaCTX) over a 24-h period in premenopausal women, elderly postmenopausal women, and elderly men. METHODS: Hourly peripheral venous blood samples were obtained from 18 healthy non-osteoporotic volunteers: premenopausal women (n = 6; mean age, 30.2 +/- 2.2 yr), postmenopausal women (n = 6; mean age, 68.2 +/- 2.6 yr), and elderly men (n = 6; mean age, 68.2 +/- 2.3 yr). Plasma PTH (1-84), OPG, betaCTX, and calcium were measured on all samples. Cosinor analysis was performed to analyze the circadian rhythm parameters. Cross-correlation analysis was used to determine the relationship between the time series of the variables. RESULTS: The 24-h mean PTH, OPG, and betaCTX concentrations were significantly higher in postmenopausal women as compared with premenopausal women and elderly men (P < 0.001). Significant circadian rhythms were observed for PTH (P < 0.05), OPG (P < 0.05), and betaCTX (P < 0.001) in all subjects. PTH secretion was characterized by two peaks in premenopausal women and elderly men and by a sustained increase in PTH concentration in postmenopausal women. OPG secretion was circadian with a daytime increase and nocturnal decrease, and a greater percent decrease in OPG secretion was observed in the postmenopausal women between 1600 and 2400 h. OPG secretion was inversely related to PTH (r = -0.4) and betaCTX (r = -0.6) secretion over a 24-h period. CONCLUSION: This report confirms a circadian rhythm for circulating OPG. The nocturnal decline in circulating OPG is greater in postmenopausal women as compared with premenopausal women and elderly men. Altered PTH secretion may contribute to the OPG secretory pattern in postmenopausal women resulting in increased nocturnal bone resorption.

Lack of an acute effect of ghrelin on markers of bone turnover in healthy controls and post-gastrectomy subjects.

BACKGROUND: Ghrelin is a gut-brain peptide that powerfully stimulates appetite and growth hormone secretion and is also known to directly regulate osteoblast cell function in vitro and in animal models. Little is known about the effects of ghrelin on bone turnover in humans. As the stomach is the main site of ghrelin synthesis, gastrectomy patients are deficient in ghrelin; they are also prone to osteopenia and osteomalacia. HYPOTHESIS: Ghrelin may play a role in bone regulation in humans; ghrelin deficiency following gastrectomy is associated with the disrupted regulation of bone turnover seen in these subjects. SUBJECTS AND METHODS: In a randomised, double-blind, placebo-controlled study 8 healthy controls and 8 post-gastrectomy subjects were infused with intravenous ghrelin (5 pmol/kg/min) or saline over 240 min on different days. Subjects were given a fixed energy meal during the infusion. Ghrelin, GH, type-1 collagen beta C-telopeptide (betaCTX), a marker of bone resorption, and procollagen type-1 amino-terminal propeptide (P1NP), a marker of bone formation, were measured. RESULTS: Fasting ghrelin was significantly lower in the gastrectomy group during the saline infusion (226.1+/-62.0 vs. 762+/-71.1 ng/l p<0.001). Growth hormone was significantly higher at 90 min after the ghrelin infusion, compared to saline in both healthy controls (61.1+/-8.8 vs. 1.4+/-0.6 mIU/l p<0.001) and gastrectomy subjects (61.1+/-11.8 vs. 0.9+/-0.2 mIU/l p<0.001) confirming the ghrelin was bioactive. Gastrectomy subjects were significantly older and had significantly higher plasma betaCTX than healthy controls at all time points (ANOVA p=0.009). After adjustment for age and BMI ghrelin was found to be a significant predictor of baseline plasma betaCTX and was inversely correlated with baseline plasma betaCTX (beta=-0.54 p=0.03 R2=26%). However, there was no significant effect of the ghrelin infusion on plasma betaCTX or P1NP in either subject group. CONCLUSIONS: Ghrelin infusion has no acute effect on markers of bone turnover in healthy controls and post-gastrectomy subjects, but is inversely correlated with bone resorption.

Effect of active acromegaly and its treatment on parathyroid circadian rhythmicity and parathyroid target-organ sensitivity.

CONTEXT: Patients with active acromegaly have increased bone turnover and skeletal abnormalities. Biochemical cure of acromegaly may represent a functional GH-deficient state and result in cortical bone loss. Reduced PTH target-organ sensitivity occurs in adult GH deficiency and may underlie the associated development of osteoporosis. OBJECTIVE: We examined the effect of active and treated acromegaly on PTH concentration and target-organ sensitivity. PATIENTS: Ten active acromegalic subjects (GH nadir > 0.3 mug/liter after 75-g oral glucose load and IGF-I above age-related reference range) and 10 matched controls participated in the study. DESIGN: Half-hourly blood and 3-h urine samples were collected on patients and controls for 24 h. Samples were analyzed for PTH, calcium (Ca), nephrogenous cAMP (NcAMP, a marker of PTH renal activity), beta C-telopeptide (bone resorption marker), and procollagen type-I amino-terminal propeptide (bone formation marker). Serum calcium was adjusted for albumin (ACa). Eight acromegalic subjects who achieved biochemical cure (GH nadir < 0.3 mug/liter after 75-g oral glucose load and IGF-I within reference range) after standard surgical and/or medical treatment reattended and the protocol repeated. RESULTS: Active acromegalic subjects had higher 24-h mean PTH, NcAMP, ACa, urine Ca, beta C-telopeptide, and procollagen type I amino-terminal propeptide (P < 0.05), compared with controls. Twenty-four-hour mean PTH increased (P < 0.001) in the acromegalic subjects after treatment, whereas NcAMP and ACa decreased (P < 0.05). CONCLUSION: Increased bone turnover associated with active acromegaly may result from increased PTH concentration and action. Biochemical cure of acromegaly results in reduced PTH target-organ sensitivity indicated by increased PTH with decreased NcAMP and ACa concentrations. PTH target-organ sensitivity does not appear to return to normal after successful treatment of acromegaly in the short term and may reflect functional GH deficiency.

Dnmt3a regulates myeloproliferation and liver-specific expansion of hematopoietic stem and progenitor cells.

DNA methyltransferase 3A (DNMT3A) mutations are observed in myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Transplantation studies have elucidated an important role for Dnmt3a in stem cell self-renewal and in myeloid differentiation. Here, we investigated the impact of conditional hematopoietic Dnmt3a loss on disease phenotype in primary mice. Mx1-Cre-mediated Dnmt3a ablation led to the development of a lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by peripheral cytopenias and by marked, progressive hepatomegaly. We detected expanded stem/progenitor populations in the liver of Dnmt3a-ablated mice. The MDS/MPN induced by Dnmt3a ablation was transplantable, including the marked hepatomegaly. Homing studies showed that Dnmt3a-deleted bone marrow cells preferentially migrated to the liver. Gene expression and DNA methylation analyses of progenitor cell populations identified differential regulation of hematopoietic regulatory pathways, including fetal liver hematopoiesis transcriptional programs. These data demonstrate that Dnmt3a ablation in the hematopoietic system leads to myeloid transformation in vivo, with cell-autonomous aberrant tissue tropism and marked extramedullary hematopoiesis (EMH) with liver involvement. Hence, in addition to the established role of Dnmt3a in regulating self-renewal, Dnmt3a regulates tissue tropism and limits myeloid progenitor expansion in vivo.

Temperature and viscosity dependence of the electron-transfer reaction between plastocyanin and cytochrome c labeled with a ruthenium(II) bipyridine complex.

The temperature and viscosity dependence of the photo-induced electron-transfer reaction between plastocyanin and cytochrome c labeled at Lys13 with Ru(4,4'-dicarboxybipyridine)(bipyridine)(2+)2 have been investigated. In these studies, a short pulse of 450 nm light was used to excite the ruthenium complex which was oxidatively quenched by the iron center of cytochrome c. The resulting Fe(II) cytochrome c was then rapidly reoxidized by plastocyanin. The reactions were investigated over a temperature range of 3.5 to 37 degrees C under low ionic strength conditions such that protein/protein complex formation was favored. The enthalpy of activation was 7 kcal mol-1 and the entropy of activation was -20 cal mol-1 K-1. Increasing the viscosity by the addition of sucrose up to 70% resulted in a 4-fold decrease in the rate constant for electron transfer. The overall results suggest a rate-limiting step that involves either dissociation of the dominant protein/protein complex or surface diffusion of the associated proteins.

Growth hormone replacement is important for the restoration of parathyroid hormone sensitivity and improvement in bone metabolism in older adult growth hormone-deficient patients.

Alterations in PTH circadian rhythm and PTH target-organ sensitivity exist in adult GH-deficient (AGHD) patients and may underlie the pathogenesis of AGHD-related osteoporosis. GH replacement (GHR) results in increased bone mineral density, but its benefit in AGHD patients over 60 yr old has been debated. To examine the effect of age on changes in PTH circadian rhythm and target-organ sensitivity after GHR, we recruited 22 AGHD patients (12 were <60 yr of age, and 10 were >60 yr of age). Half-hourly blood samples were collected for PTH, calcium, phosphate, nephrogenous cAMP (marker of renal PTH activity), type-I collagenbeta C-telopeptide (bone resorption marker), and procollagen type-I amino-terminal propeptide (bone formation marker) before and after 1, 3, 6, and 12 months of treatment with GHR. Significant PTH circadian rhythms were present in both age groups throughout the study. After GHR, PTH decreased and nephrogenous cAMP, adjusted calcium, and bone turnover markers increased in both groups, suggesting increased PTH target-organ sensitivity. In younger patients, the changes were significant after 1 month of GHR, but, in older patients, the changes were delayed until 3 months, with maximal changes at 12 months. Older AGHD patients derive benefit from GHR in terms of improvement in PTH sensitivity and bone metabolism. Their response appears delayed and may explain why previous studies have not shown a positive effect of GHR on bone mineral density in older AGHD patients.

A detailed assessment of alterations in bone turnover, calcium homeostasis, and bone density in normal pregnancy.

The effects of pregnancy on bone turnover and the potential risk of developing an osteoporotic fracture in pregnancy are controversial. Utilizing biochemical markers of bone formation and resorption and dual-energy X-ray absorptiometry (DEXA), bone turnover before, during, and after pregnancy was studied in detail. Ten women (mean age 30 years; range 23-40) were recruited. Prepregnancy data were obtained and then a review was performed at 2-week intervals , once pregnancy was confirmed, until 14 weeks of gestation and thereafter monthly until term. Bone mineral density (BMD) was estimated by DEXA scanning of hip, spine, and forearm preconception and postpartum. In addition, BMD of the forearm at 14 weeks and 28 weeks gestation was obtained. All pregnancies had a successful outcome. Urinary free pyridinium cross-links, free pyridinoline (fPyr) and free deoxypyridinoline (fDPyr), were normal prepregnancy (mean [+/-SD]) 14.6 nmol/mmol (1.8) and 5.0 nmol/mmol (1.0) creat, respectively. By 14 weeks, they had increased to 20.8 nmol/mmol (4.3) and 6.1 nmol mmol (1.4) (both p < 0.02) and by 28 weeks to 26.3 nmol/mmol (5.6) and 7.4 nmol/mmol (1.6) (both p < 0.01). The ratio of fPyr to fDPyr remained constant. A similar significant increase was observed in N-telopeptide (NTx). Bone formation was assessed by measurement of carboxyterminal propeptide of type 1 collagen (P1CP) and bone-specific alkaline phosphatase (BSAP). Neither were altered significantly before 28 weeks, but subsequently mean P1CP increased from 110 microg/liter (23) to 235 microg/liter (84) at 38 weeks and mean BSAP increased from 11.1 U/liter (5.0) to 28.6 U/liter (11.1) (p < 0.01 for both variables). Lumbar spine (L1-L4) BMD decreased from a prepregnancy mean of 1.075 g/cm (0.115) to 1.054 g/cm2 (0.150) postpartum (p < 0.05). Total hip BMD decreased from a prepregnancy mean of 0.976 g/cm2 (0.089) to 0.941 g/cm2 (0.097) (p < 0.05). Forearm BMD at midradius, one-third distal and ultradistal decreased but did not reach statistical significance. As assessed by these bone markers, in the first 2 trimesters of pregnancy, bone remodeling is uncoupled with a marked increase in bone resorption. A corresponding increase in formation markers is not observed until the third trimester. Spinal BMD exhibits a significant decrease from prepregnancy to the immediate postpartum period with a mean reduction in BMD of 3.5 % in 9 months.

Dietary fish oil reduces basal and ultraviolet B-generated PGE2 levels in skin and increases the threshold to provocation of polymorphic light eruption.

The sunburn response is markedly reduced by dietary fish oil rich in omega-3 polyunsaturated fatty acids. Because prostaglandins mediate the vasodilatation, we examined the effect of fish oil on ultraviolet (UV) B-induced prostaglandin metabolism. In addition we assessed the potential photoprotective effect of fish oil in light-sensitive patients. Thirteen patients with polymorphic light eruption received dietary supplements of fish oil rich in omega-3 polyunsaturated fatty acids for 3 months. At baseline and 3 months, the minimal erythema dose of UVB irradiation was determined, and a graded UVA challenge given to a forearm to assess the threshold dose for papule provocation. Suction blisters were raised on the other forearm, on control skin, and on skin irradiated with four times the minimal erythema dose of UVB 24 h previously, and blister fluid prostaglandin E2 was measured by radioimmunoassay. Following 3 months of fish oil, the mean minimal erythema dose of UVB irradiation increased from 19.8 +/- 2.6 to 33.8 +/- 3.7 mJ/cm2 (mean +/- SEM), p < 0.01. The UVA provocation test was positive in 10 patients at baseline, and after 3 months nine of these showed reduced sensitivity to papule provocation, p < 0.001. Before fish oil, PGE2 increased from 8.6 (SEM 2.1) ng/ml in control skin to 27.2 (11) ng/ml after UVB, p < 0.01. Following 3 months of fish oil, PGE2 decreased to 4.1 (1) and 9.6 (2.4) ng/ml in control and irradiated skin, respectively, p < 0.05. Reduction of UV-induced inflammation by fish oil may be due, at least partially, to lowered prostaglandin E2 levels. The photoprotection against UVA-provocation of a papular response suggests a clinical application for fish oil in polymorphic light eruption.

Parathyroid hormone secretory pattern, circulating activity, and effect on bone turnover in adult growth hormone deficiency.

Adult growth hormone deficiency (AGHD) is associated with osteoporosis. Reports have associated parathyroid hormone (PTH) circadian rhythm abnormalities with osteoporosis. Furthermore, there is evidence of relative PTH insensitivity in AGHD patients. Factors regulating PTH circadian rhythm are not fully understood. There is evidence that serum phosphate is a likely determinant of PTH rhythm. The aim of this study was to investigate PTH circadian rhythm and its circulating activity and association with bone turnover in untreated AGHD patients compared to healthy individuals. We sampled peripheral venous blood at 30-min and urine at 3-h intervals during the day over a 24-h period from 1400 h in 14 untreated AGHD patients (7 M, 7 W; mean age, 49.5 +/- 10.7 years) and 14 age (48.6 +/- 11.4 years; P = NS) and gender-matched controls. Cosinor analysis was performed to analyze rhythm parameters. Cross-correlational analysis was used to determine the relationship between variables. Serum PTH (1-84), phosphate, total calcium, urea, creatinine, albumin, type I collagen C-telopeptides (CT(x)), a bone resorption marker, and procollagen type I amino-terminal propeptide (PINP), a bone formation marker, were measured on all samples. Nephrogenous cyclic adenosine monophosphate (NcAMP), which reflects the renal activity of PTH, was calculated from plasma and urinary cAMP. Urinary calcium and phosphate were measured on all urine samples. Significant circadian rhythms were observed for serum PTH, phosphate, CT(x), and PINP in AGHD and healthy subjects (P < 0.001). No significant rhythm was observed for serum-adjusted calcium. PTH MESOR (rhythm-adjusted mean) was significantly higher (P < 0.05), whereas the MESOR values for phosphate, CT(x) (P < 0.05), and PINP (P < 0.001) were lower in AGHD patients than in controls. AGHD patients had significantly lower 24-h NcAMP (P < 0.001) and higher urinary calcium excretion (P < 0.05). Maximum cross-correlation between PTH and phosphate (r = 0.75) was observed when PTH was lagged by 1.5 h in healthy individuals, suggesting that changes in phosphate precede changes in PTH concentration. PTH/CT(x) and PTH/PINP showed maximum correlation when CT(x) (r = 0.68) and PINP (r = 0.71) were lagged by 3 h. In AGHD patients, compared to controls the maximum correlation between PTH/phosphate (r = 0.88, P = 0.007), PTH/CTx (r = 0.61, P = 0.027), and PTH/PINP (r = 0.65, P = 0.028) was observed when the lag time was reduced by 1.5 h in all variables, with changes in PTH and phosphate occurring at concurrent time points. Our data suggest decreased end-organ sensitivity to the effects of PTH in AGHD patients, resulting in a significantly lower NcAMP, low bone turnover, and higher calcium excretion in the presence of significantly higher PTH concentrations. We have also demonstrated that changes in serum phosphate precede those of PTH, which in turn precede changes in bone resorption and formation in healthy individuals. This relationship was altered in AGHD patients. These results suggest a possible role for GH in regulating PTH secretion and the bone remodeling process.

Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase.

A new technique has been introduced to measure interprotein electron transfer which involves photoexcitation of a tris(bipyridine)ruthenium (Ru) complex covalently attached to one of the proteins. Four different strategies have been developed to specifically attach Ru to protein lysine amino groups, histidine imidazole groups, and cysteine sulhydryl groups. These strategies have been used to prepare more than 20 different singly-labeled Ru-cytochrome c derivatives. The new ruthenium photoexcitation technique has been used to study the mechanism for electron transfer between cytochrome c and cytochrome c peroxidase. Laser excitation of a complex between Ru-cytochrome c and cytochrome c peroxidase compound I results in formation of Ru(II*) which is a strong reducing agent, and rapidly transfers an electron to heme c Fe(III) to form Fe(II). The heme c Fe(II) then rapidly transfers an electron to the Trp-191 radical cation in CMPI. The rate constant for this reaction is 6 x 10(4) s-1 for a horse Ru-cytochrome c derivative labeled at lysine 27, and greater than 10(6) s-1 for yeast Ru-cytochrome c derivatives. A second laser flash results in electron transfer from heme c to the oxyferryl heme in cytochrome c peroxidase compound II with a rate constant of 350 s-1. The ruthenium photoreduction technique has been used to study the interaction domain between the two proteins, the pathway for electron transfer to the radical cation and the oxyferryl heme, and the specific residues in the heme crevice which control the electron transfer properties of the Trp-191 radical cation and the oxyferryl heme.

Serum cystyl aminopeptidase activity in the 36th week of pregnancy.

Serum cystyl aminopeptidase (CAS) activity was estimated at 36 weeks' gestation in 209 normotensive pregnancies. The highest activity was found in 31 women who had spontaneous deliveries before 38 weeks' gestation and the lowest in 76 women who were induced after term. The enzyme levels in 117 women who developed hypertension of pregnancy were higher than for normotensives; the highest levels were found in 32 women with pre-eclampsia. A correlation was found between serum CAS activity at 36 weeks' gestation and the birth weight of babies of women who went into spontaneous labour at term (277 to 283 days' gestation).

Change in plasma cystyl aminopeptidase (oxytocinase) between 30-34 weeks' gestation as a predictor of pregnancy-induced hypertension.

Ninety-two primigravidas were screened biweekly by measurement of plasma cystyl aminopeptidase from 28 weeks' gestation until delivery. Fourteen developed hypertension with or without proteinuria after 36 weeks. The hypertensive group had significantly higher levels of the enzyme at 30 weeks, although this difference was not significant at 34 weeks. The rise in the hypertensive group was less than 50% between weeks 30-34 in all cases, whereas it was over 50% in all but two of the 43 controls. The difference in the rates of increase of the enzyme and its action on antidiuretic hormone may have some bearing on the subsequent development of hypertension.

Measurement of plasma 1,25 dihydroxyvitamin D using a novel immunoextraction technique and immunoassay with iodine labelled vitamin D tracer.

We evaluated a novel assay for the measurement of 1,25 dihydroxyvitamin D (1,25 (OH)2D). Immunoextraction of 1,25(OH)2D is performed using a mini column containing a solid-phase monoclonal antibody followed by radioimmunoassay (RIA) using an 125I-labelled 1,25(OH)2D derivative tracer and Sac-cell separation. The mean recovery of 1,25(OH)2D3 was 101%, linearity was excellent, inter- and intra-assay coefficients of variation were 9, 8 and 13% and 11, 10 and 14% at low, medium and high concentrations of 1,25 (OH)2D3, respectively. The cross-reactivity of vitamin D metabolites was < 0.0015% for 25-hydroxyvitamin D3, 24, 25 dihydroxyvitamin D3 and dihydrotachysterol and 0.54% for 1 alpha calcidol. 1,25 dihydroxyvitamin D2 cross-reactivity was 79%. The detection limit of the assay was 5 pmol/L. Comparison with a commercial radio receptor assay (RRA) and an in-house RIA gave regression equations of y = 0.94x + 11.8 (r = 0.98) and y = 0.91x-1.7 (r = 0.95), respectively, with no major discrepancies between the methods in all patient groups studied. Plasma concentrations of 1,25(OH)2D obtained with the assay were as follows: normal, unsupplemented subjects: mean 88, range 48-155 pmol/L, n = 68, patients with chronic renal failure: mean 11, range 3-36 pmol/L, n = 27, primary hyperparathyroidism: mean 198, range 130-299 pmol/L, n = 23, Paget's disease: mean 92, range 42-149 pmol/L, n = 24, osteomalacia: mean 43, range 27-61 pmol/L, n = 9. A minimum sample volume of 300 microL is required, the hands-on time is significantly less than other commercial assays and the measuring procedure is gamma counting rather than scintillation counting. The assay offers several advantages over previous methods and should allow more laboratories to offer measurement of 1,25(OH)2D as part of their repertoire.

Evaluation of three methods of protein analysis for serum and heart homogenates.

The Biuret, Ultraviolet and Lowry techniques for the determination of total protein content were compared in 31 samples of patient serum and in 30 rat heart tissue homogenates. Statistically significant differences were found to exist between values obtained with each method. Human serum and heart homogenate protein were both lowest when assayed by the Lowry procedure, while serum and homogenate values were highest with the Ultraviolet and Biuret method, respectively. Possible sources of these discrepancies are discussed. Although the Biuret method may not be as sensitive as the other two methods, it proved to be the most reproducible technique for determining total protein concentration in the particular types of samples used in this study.

Are manual gestures, verbal descriptors and pain radiation as reported by patients reliable indicators of myocardial infarction? Preliminary findings and implications.

Patients experiencing an episode of acute chest pain need to be assessed promptly and effectively to ensure optimal management. The aim of this study was to investigate whether there were specific aspects of patients' symptom reports which could be viewed as additional indicators of myocardial infarction (MI) and contribute to the assessment process. The sample consisted of 267 patients who presented with an episode of acute chest pain. Methods of data collection were based on, or modified from, previous studies which had investigated the use of manual gestures, choice of verbal descriptors and extent of pain radiation in patients with and without MI. To determine whether these variables were of significance in the diagnosis, the responses of those with (n = 118) and without MI (n = 149) were compared. The results suggest that it is currently impossible to draw any conclusions as to whether the variables studied can be judged as reliable indicators of MI. The findings indicate that there are some differences between the groups particularly in the language used. Moreover, women with MI characterized their symptoms through stronger emotive words such as 'worrying' (P = 0.014) 'frightening' and 'intolerable' and also differed from their male counterparts in their reports regarding pain radiation. Implications for practice and for research are discussed.