Post-diagnosis serum 25-hydroxyvitamin D concentrations in women treated for breast cancer participating in a lifestyle trial in Italy.

OBJECTIVE: To report cross-sectionally serum levels of 25-hydroxyvitamin D [25(OH)D] in women living in Italy within 12 months from breast cancer (BC) diagnosis. METHODS: Baseline data were obtained from 394 women diagnosed with primary BC, enrolled from 2016 to 2019 in a lifestyle trial conducted in Italy. Subjects' characteristics were compared between two 25(OH)D concentrations (hypovitaminosis D<20 and ≥20 ng/mL) with the Chi-squared test or Fisher's exact test for small-expected counts. Using multiple logistic regression-adjusted models, we estimated odds ratios (ORs) of hypovitaminosis D with 95% confidence intervals (CIs) in the total sample and in the unsupplemented subgroup. RESULTS: Hypovitaminosis D was found in 39% of all subjects, 60% in unsupplemented subjects, and 10% in supplemented subjects. Increasing ORs of hypovitaminosis D were found with increasing body mass index, 25-30, >30, and ≥35 versus <25 kg/m2 (ORs: 2.50, 4.64, and 5.81, respectively, in the total cohort and ORs: 2.68, 5.38, and 7.08 in the unsupplemented); living in the most southern Italian region (OR 2.50, 95%CI 1.22-5.13); and with hypertriglyceridemia (OR 2.46; 95%CI 1.16-5.22), chemotherapy history (OR 1.86, 95%CI 1.03-3.38), and inversely with anti-estrogenic therapy (OR 0.43, 95%CI 0.24-0.75) in the total sample. CONCLUSIONS: Hypovitaminosis D in women recently diagnosed with BC and participating in a lifestyle trial in Italy was widespread and highest with obesity, hypertriglyceridemia, and chemotherapy use. Considering that hypovitaminosis D is a risk factor for lower efficacy of bone density treatments and possibly BC mortality, our results suggest the need to promptly address and treat vitamin D deficiency.

Understanding the risk of emerging bacterial resistance to over the counter antibiotics in topical sore throat medicines.

AIMS: The aims of this study were to explore the development of bacterial resistance and cross-resistance in four common human pathogens following realistic exposure to antibiotics found in over-the-counter (OTC) sore throat medicines: gramicidin, neomycin, bacitracin and tyrothricin. METHODS AND RESULTS: Bacterial exposure to in-use (concentration in the product before use) and diluted concentration (i.e. during use) of antibiotic where conducted in broth for 24 h or until growth was visible. The changes in bacterial susceptibility profile before and after exposure was determined using standardized ISO microdilution broth. Antibiotic testing was performed according to EUCAST guidelines. We demonstrated that test bacteria were able to survive exposure to the in-use concentrations of some antibiotics used in OTC medicines. Exposure to during use concentrations of bacitracin resulted in stable increase in minimal inhibitory concentration (MIC) (>8-fold) in Staphylococcus aureus and Acinetobacter baumannii. Exposure to tyrothricin resulted in a stable increase in MIC (2·4-fold) in Klebsiella pneumoniae, and exposure to neomycin resulted in a stable increase MIC (5000-fold higher than the baseline) in Streptococcus pyogenes. Clinical cross-resistance to other antibiotics (ciprofloxacin, fusidic acid, gentamicin, cefpodoxime, amoxicillin/clavulanic acid and cefotaxime) was also demonstrated following exposure to bacitracin or tyrothricin. Bacitracin exposure lead to a stable bacterial resistance after 10 passages. CONCLUSIONS: Our results indicate that OTC antibiotic medicines have the potential to drive resistance and cross-resistance in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Tackling antibiotic resistance is a high worldwide priority. It is widely accepted that the overuse and misuse of antibiotics increase the risk of the development and spread of antibiotic resistance within communities. A number of OTC sore throat products, widely available across the world for topical use in respiratory indications, contain locally delivered antibiotics. Our findings showed that these antibiotics in OTC medicines present a risk for emerging cross-resistance in a number of bacterial respiratory pathogens.

Scale-dependent patterns of intraspecific trait variations in two globally invasive species.

Animal species often show substantial intraspecific trait variability (ITV), yet evidence for its flexibility across multiple ecological scales remains poorly explored. Gaining this knowledge is essential to better understand the different processes maintaining ITV in nature. Due to their broad geographic ranges, widespread invasive species are expected to display strong phenotypic variations across their distribution. Here, we quantified the scale-dependent patterns of morphological variability among invasive populations of two global freshwater invaders-red swamp crayfish Procambarus clarkii and pumpkinseed sunfish Lepomis gibbosus-both established in American and European lakes. We quantified patterns in body morphology across different ecological (Individual and Population) and spatial scales (Region). We then analyzed the scale-dependency of morphological variations among lake populations that span a diversity of abiotic and biotic conditions. Next, we used stable isotope analyses to test the existence of ecomorphological patterns linking morphology and trophic niche of individuals. We found that trait variations mainly accounted for at the regional and individual levels. We showed that populations of both species strongly differed between United States and Europe whereas habitat characteristics had a relatively minor influence on morphological variations. Stable isotope analyses also revealed that ecomorphological pattern for the trophic position of L. gibbosus was region-dependent, whereas no ecomorphological patterns were observed for P. clarkii. Overall, our study strongly supports the notion that the patterns of phenotypic variability among invasive populations are likely to modulate the ecological impacts of invasive species on recipient ecosystems.

Contrasting ecological impacts of geographically close invasive populations.

Intraspecific trait variability is now well recognized as a key component of biodiversity explaining how individuals within a species can differentially interact with their environment. To date, however, this concept has rarely been incorporated in the study of biological invasions, despite its provision of new insights into invasive species management. Here, we used an experimental approach to investigate how invasive red swamp crayfish (Procambarus clarkii) derived from geographically close ecosystems can differentially impact prey community structure and relevant ecosystem processes. We also compared the magnitude of the effects induced by invasive species introduction with those induced by intraspecific variability. Our results showed that effects of intraspecific variability can be strong for direct interactions such as resource (e.g., leaf litter, snails) consumption and of similar magnitude to the effects induced by the introduction of the invasive species when considering indirect interactions (e.g., primary production, ecosystem metabolism). Overall, these results highlighted that invasive populations of the same species are not ecologically equivalent, with each population acting differently on their recipient ecosystem.

Honeybee navigation: critically examining the role of the polarization compass.

Although it is widely accepted that honeybees use the polarized-light pattern of the sky as a compass for navigation, there is little direct evidence that this information is actually sensed during flight. Here, we ask whether flying bees can obtain compass cues derived purely from polarized light, and communicate this information to their nest-mates through the 'waggle dance'. Bees, from an observation hive with vertically oriented honeycombs, were trained to fly to a food source at the end of a tunnel, which provided overhead illumination that was polarized either parallel to the axis of the tunnel, or perpendicular to it. When the illumination was transversely polarized, bees danced in a predominantly vertical direction with waggles occurring equally frequently in the upward or the downward direction. They were thus using the polarized-light information to signal the two possible directions in which they could have flown in natural outdoor flight: either directly towards the sun, or directly away from it. When the illumination was axially polarized, the bees danced in a predominantly horizontal direction with waggles directed either to the left or the right, indicating that they could have flown in an azimuthal direction that was 90° to the right or to the left of the sun, respectively. When the first half of the tunnel provided axial illumination and the second half transverse illumination, bees danced along all of the four principal diagonal directions, which represent four equally likely locations of the food source based on the polarized-light information that they had acquired during their journey. We conclude that flying bees are capable of obtaining and signalling compass information that is derived purely from polarized light. Furthermore, they deal with the directional ambiguity that is inherent in polarized light by signalling all of the possible locations of the food source in their dances, thus maximizing the chances of recruitment to it.

Honeybee navigation: following routes using polarized-light cues.

While it is generally accepted that honeybees (Apis mellifera) are capable of using the pattern of polarized light in the sky to navigate to a food source, there is little or no direct behavioural evidence that they actually do so. We have examined whether bees can be trained to find their way through a maze composed of four interconnected tunnels, by using directional information provided by polarized light illumination from the ceilings of the tunnels. The results show that bees can learn this task, thus demonstrating directly, and for the first time, that bees are indeed capable of using the polarized-light information in the sky as a compass to steer their way to a food source.

The moment before touchdown: landing manoeuvres of the honeybee Apis mellifera.

Although landing is a crucial part of insect flight, it has attracted relatively little study. Here, we investigate, for the first time, the final moments of a honeybee's (Apis mellifera) landing manoeuvre. Using high-speed video recordings, we analyse the behaviour of bees as they approach and land on surfaces of various orientations. The bees enter a stable hover phase, immediately prior to touchdown. We have quantified behaviour during this hover phase and examined whether it changes as the tilt of the landing surface is varied from horizontal (floor), through sloped (uphill) and vertical (wall), to inverted (ceiling). The bees hover at a remarkably constant distance from the surface, irrespective of its tilt. Body inclination increases progressively as the tilt of the surface is increased, and is accompanied by an elevation of the antennae. The tight correlation between the tilt of the surface, and the orientation of the body and the antennae, indicates that the bee's visual system is capable of inferring the tilt of the surface, and pointing the antennae toward it. Touchdown is initiated by extending the appendage closest to the surface, namely, the hind legs when landing on horizontal or sloping surfaces, and the front legs or antennae when landing on vertical surfaces. Touchdown on inverted surfaces is most likely triggered by a mechanosensory signal from the antennae. Evidently, bees use a landing strategy that is flexibly tailored to the varying topography of the terrain.

Identificação das queixas de adultos separados atendidos em uma clínica-escola de psicologia / Complaints identified in separated adults attended in a psychology school-clinic

Este estudo objetivou caracterizar as queixas dos adultos separados atendidos em uma clínica-escola da cidade de São Paulo, entre 1996 e 2000, em função do sexo, faixa etária, escolaridade, tipos de queixas, encaminhamento e adesão ao tratamento. Empregou-se uma metodologia descritiva retrospectiva documental, de consulta aos prontuários clínicos. Dos 28 pacientes separados atendidos nesse período, encontrou-se a predominância do sexo feminino (78,5

), na faixa etária de 31 a 35 anos (28,5

), 2º grau completo (25

), com encaminhamento realizado pelo psicólogo ou estudante de Psicologia (35,7

). Foram listadas 86 queixas, predominando as de ansiedade, insegurança, medo (42,8

) e depressão (32,1

). A separação foi citada como parte da história clínica em 60,7

dos casos; o encaminhamento para psicoterapia breve ocorreu em 75

dos processos, e foi concluída em 63

The research had the aim to characterize separated adults who were attended in a school-clinic of São Paulo, between 1996 and 2000, according to their gender, age group, educational level, types of complaints, prescription and adhesion to treatment. It was carried out through a retrospective descriptive documental methodology, by consulting the clinical files. Among the 28 patients examined in the period, the predominance was female (78,5

), with a group age between 31 and 35 years old (28,5

) and completed high school level (25

), prescribed by a psychologist or a Psychology student (35.7

). There were 86 complaints listed with the predominance of anxiety, insecurity, fear (42,8

) and depression (32,1

). The separation was referred to as part of the clinical history in 60,7

of the cases; the prescription of short term psychotherapy occurred in 75

of the cases and 63

dos casos. A principal implicação deste estudo refere-se à falta de pesquisas relacionadas que especifiquem as queixas das pessoas separadas ou divorciadas.(AU)

of the cases were concluded. The main implication of this paper refers to the lack of research that specifies the complaints of separated or divorced persons.(AU)

Identificação das queixas de adultos separados atendidos em uma clínica-escola de psicologia / Complaints identified in separated adults attended in a psychology school-clinic

Este estudo objetivou caracterizar as queixas dos adultos separados atendidos em uma clínica-escola da cidade de São Paulo, entre 1996 e 2000, em função do sexo, faixa etária, escolaridade, tipos de queixas, encaminhamento e adesão ao tratamento. Empregou-se uma metodologia descritiva retrospectiva documental, de consulta aos prontuários clínicos. Dos 28 pacientes separados atendidos nesse período, encontrou-se a predominância do sexo feminino (78,5 por cento), na faixa etária de 31 a 35 anos (28,5 por cento), 2º grau completo (25 por cento), com encaminhamento realizado pelo psicólogo ou estudante de Psicologia (35,7 por cento). Foram listadas 86 queixas, predominando as de ansiedade, insegurança, medo (42,8 por cento) e depressão (32,1 por cento). A separação foi citada como parte da história clínica em 60,7 por cento dos casos; o encaminhamento para psicoterapia breve ocorreu em 75 por cento dos processos, e foi concluída em 63 por cento dos casos. A principal implicação deste estudo refere-se à falta de pesquisas relacionadas que especifiquem as queixas das pessoas separadas ou divorciadas.

The research had the aim to characterize separated adults who were attended in a school-clinic of São Paulo, between 1996 and 2000, according to their gender, age group, educational level, types of complaints, prescription and adhesion to treatment. It was carried out through a retrospective descriptive documental methodology, by consulting the clinical files. Among the 28 patients examined in the period, the predominance was female (78,5 percent), with a group age between 31 and 35 years old (28,5 percent) and completed high school level (25 percent), prescribed by a psychologist or a Psychology student (35.7 percent). There were 86 complaints listed with the predominance of anxiety, insecurity, fear (42,8 percent) and depression (32,1 percent). The separation was referred to as part of the clinical history in 60,7 percent of the cases; the prescription of short term psychotherapy occurred in 75 percent of the cases and 63 percent of the cases were concluded. The main implication of this paper refers to the lack of research that specifies the complaints of separated or divorced persons.

Does initial spacing influence crown and hydraulic architecture of Eucalyptus marginata?

Long-term declines in rainfall in south-western Australia have resulted in increased interest in the hydraulic characteristics of jarrah (Eucalyptus marginata Donn ex Smith) forest established in the region's drinking water catchments on rehabilitated bauxite mining sites. We hypothesized that in jarrah forest established on rehabilitated mine sites: (1) leaf area index (L) is independent of initial tree spacing; and (2) more densely planted trees have less leaf area for the same leaf mass, or the same sapwood area, and have denser sapwood. Initial stand densities ranged from about 600 to 9000 stems ha(-1), and trees were 18 years old at the time of sampling. Leaf area index was unaffected by initial stand density, except in the most sparsely stocked stands where L was 1.2 compared with 2.0-2.5 in stands at other spacings. The ratio of leaf area to sapwood area (A(l):A(s)) was unaffected by tree spacing or tree size and was 0.2 at 1.3 m height and 0.25 at the crown base. There were small increases in sapwood density and decreases in leaf specific area with increased spacing. Tree diameter or basal area was a better predictor of leaf area than sapwood area. At the stand scale, basal area was a good predictor of L (r(2) = 0.98, n = 15) except in the densest stands. We conclude that the hydraulic attributes of this forest type are largely independent of initial tree spacing, thus simplifying parameterization of stand and catchment water balance models.

Population-based register of acute myocardial infarction: manual of operations.

Cardiovascular disease is the leading cause of death and hospitalization in both sexes in nearly all countries of Europe. The main forms of cardiovascular disease are ischaemic heart disease and stroke. The magnitude of the problem contrasts with the shortage, weak quality and comparability of data available in most European countries. Innovations in medical, invasive and biological treatments have substantially contributed to the escalating costs of health services. It is therefore urgent to obtain reliable information on the magnitude and distribution of the disease for both adequate health planning (including preventive strategies) and clinical decision making with correct cost-benefit assessments.A stepwise surveillance procedure based on standardized data collection, appropriate record linkage and validation methods was set up by the EUROCISS Project (EUROpean Cardiovascular Indicators Surveillance Set) to build up comparable and reliable indicators (attack rate and case fatality) for the surveillance of acute myocardial infarction/acute coronary syndrome at population level. This manual of operations is intended for health professionals and policy makers and provides a standardized and simple model for the implementation of a population-based register. It recommends to start from a minimum data set and then follow a stepwise procedure. Before implementing a population-based register, it is important to identify the target population under surveillance which should preferably cover a well-defined geographical and administrative area or region representative of the whole country for which population data and vital statistics (mortality and hospital discharge records at minimum) are routinely collected and easily available each year. All cases among residents should be recorded even if the case occurs outside the area. Validation of a sample of fatal and nonfatal events is mandatory.

Acid-base transport in Henle's loop: the effects of reduced renal mass and diabetes.

The loop of Henle (LOH) is an important site of renal acidification. Using the in vivo microperfusion technique of LOH combined with quantitative polymerase chain reaction (PCR) performed on isolated thick ascending limbs (TAL), we demonstrated that the Na + -H + exchanger is the main transport mechanism involved, although a small, but significant contribution from the H+-ATPase also occurs. Among the various Na+-H+ exchanger isoforms we have evidenced that NHE3 is expressed and functionally active along the TAL. Since the LOH is exposed to osmotic stress, bicarbonate transport was also measured under medullary hypotonicity conditions, which led to the stimulation of bicarbonate reabsorption. We demonstrated that the LOH can participate in the tubular adaptation to an increased filtered bicarbonate load by increasing net LOH bicarbonate transport. In this setting, at the molecular level, mRNA and protein abundance of NHE3 were also stimulated, and coincided with an increase in NHE3 activity. Finally, NHE3 expression and abundance was highly stimulated in the early phase of diabetes, which is characterized by increased glomerular filtration rate (GFR).

[Glomerulo-tubular balance in diabetes mellitus: molecular evidence and clinical consequences]. / Bilancio glomerulo-tubulare nel diabete mellito: evidenze molecolari e conseguenze cliniche.

Diabetes mellitus is fast becoming a world epidemic. About one-third of individuals with diabetes, after 10 yrs, develop diabetic nephropathy, the first cause of end-stage kidney disease. The evolution of diabetic nephropathy can be considered in three stages: glomerular hyperfiltration, microalbuminuria (30-300 mg/24 hr) and proteinuria (>300 mg/24 hr). This study was designed to investigate the tubular basis of glomerular hyperfiltration in early diabetes mellitus. Diabetes was inducted in rats with i.p. streptozotocin (65 mg/kg bw) for 6 days. At the end of the treatment, the glomerular filtration rate (GFR), measured by inulin clearance, had substantially increased in diabetic rats compared with controls. Quantitative polymerase chain reaction (PCR) and Western blot analysis reveal that in diabetic rats compared with controls, mRNA and protein abundance was higher for type 3 sodium/hydrogen exchanger (NHE3) in proximal tubule and ascending limbs of Henle's loop, and higher for bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2) in ascending limbs of Henle's loop. Western blot analysis confirmed the PCR results. Finally, the abundance of á -ENaC protein was unchanged in diabetic rats compared to controls. These results show that the primary sodium reabsorption increase in proximal tubule reduces salt concentrations at the macula densa. This elicits a tubuloglomerular feedback-dependent increase in single nephron GFR.

Morphoquantitative aspects of NADH-diaphorase myenteric neurons in the ileum of diabetic rats treated with acetyl-L-carnitine.

In this work, we investigated the effect of the acetyl-L-carnitine (ALC) supplementation (200 mg/kg/day) on the myenteric neurons of the ileum of rats made diabetic by streptozotocin (35 mg/kg, i.v.). Four groups were used: diabetic (D), diabetic supplemented with ALC (DC), control (C) and control supplemented with ALC (CC). After 15 weeks of diabetes induction the animals were killed and the ileum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The density of neurons seen in 12.72 mm2 of ileum showed no difference among the groups, although in group D it was 22% smaller than in group C, while group DC was 9% smaller to group CC. The profiles of the cell bodies (PC) of 1000 neurons per group were analysed. The neurons PC in group D decreased (P < 0.0001) when compared with other groups and increased (P < 0.0001) when compared with group DC. The incidence of neurons with a PC inferior to 200 microm2 was larger in group D. The frequency of neurons with a PC higher than 200 microm2 in group DC was close to those seen in groups C and CC. We concluded that ALC eases the loss of neurons and makes the incidence of myenteric neurons with a PC higher than 200 microm2 similar to the control rats.

Causes of Cheyne-Stokes respiration.

Cheyne-Stokes respiration (CSR) is one of several types of unusual breathing with recurrent apneas (dysrhythmias). Reported initially in patients with heart failure or stroke, it was then recognized both in other diseases and as a component of the sleep apnea syndrome. CSR is potentiated and perpetuated by changing states of arousal that occur during sleep. The recurrent hypoxia and surges of sympathetic activity that often occur during the apneas may have serious health consequences. Heart failure and stroke are risk factors for sleep apnea. The recurrent apneas and intermittent hypoxia occurring with sleep apnea further damage the heart and brain. Although all breathing dysrhythmias do not have the same cause, instability in the feedback control involved in the chemical regulation of breathing is the leading cause of CSR. Mathematical models have helped greatly in the understanding of the causes of recurrent apneas.

Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

[New concepts of tubular calcium transport in the kidney: clinical implications]. / Nuove acquisizioni ed implicazioni cliniche del trasporto del calcio a livello del tubulo renale.

The kidney plays an important role in calcium homeostasis. In this review we discuss new concepts in tubular calcium transport, related proteins and the clinical implications of these new findings. Most calcium reabsorption occurs in the proximal tubules via the passive paracellular pathway, but calcium reabsorption also occurs in the thick ascending limb of Henle's loop (50% via the transcellular pathway). Finally, at the level of the distal convoluted tubule and connecting tubule calcium is reabsorbed via the active transcellular route. The Calcium-sensing receptor, localised along the thick ascending limb of Henle's loop, regulates the urinary calcium excretion in response to changes in extracellular calcium concentration. The Epithelial Calcium Channel 1 is a highly Ca2+-sensitive channel that is predominantly present in the connecting tubule. The Calbindin D(28k) is a cytoplasmatic protein expressed in the distal tubule, it is involved both in transcellular calcium diffusion and in the control of intracellular calcium concentration. Heterozygous mutations in the gene for the calcium sensing receptor, which result in a loss of function by the receptor, are associated with familial hypocalciuric hypercalcaemia. Mutations involving a gain of function have been associated with hypocalcaemia with normal serum parathyroid hormone concentration. Bartter's syndrome, caused by a dysfunction of thick ascending limb cells, is associated with calcium wasting. On the contrary, Gitelman's syndrome, caused by a dysfunction of the distal tubule, is characterised by hypocalciuria and hypomagnesemia.

Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells.

Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets.

N-terminal PDZ domain is required for NHERF dimerization.

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.

Synthesis and characterization of insulin-like growth factor (IGF)-1 photoprobes selective for the IGF-binding proteins (IGFBPS). photoaffinity labeling of the IGF-binding domain on IGFBP-2.

Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes, N(alphaGly1)-(4-azidobenzoyl)-IGF-1 (abG(1)IGF-1) and N(alphaGly1)-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1 (bedG(1)IGF-1), selective for the IGFBPs were synthesized by derivatization of the alpha-amino group of Gly(1), known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG(1)IGF-1.recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG(1)IGF-1.rhIGFBP-2 complex revealed photoincorporation within residues 212-227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2.