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Parental factors contribute to ADHD, partly in an etiological way and partly as moderators and mediators of child outcomes and treatment effects. An important aspect of parenting seems to be parental reflective functioning (PRF), defined as the parent's capacity to reflect upon his own and his child's internal mental experience. The studies on parenting factors linked to ADHD have not extensively investigated the role of PRF. Recent findings on interventions have begun to consider mentalization to promote empathy and emotion regulation in parents, but empirical studies assessing PRF are still scarce. The aim of this cross-sectional study was to compare specific familial and parental functioning characteristic between parents of children with attention deficit/hyperactivity disorder (ADHD) and parents of controls without ADHD. A clinical sample of 41 children with ADHD aged 8-11 years and their parents was compared with a matched, non-clinical sample of 40 children. Three aspects of parental functioning were investigated: parental symptomatology, parental alliances and PRF; children's differences in strength and difficulty profiles were also assessed. The results showed that families of children with ADHD had lower socioeconomic status, and both mothers and fathers of the same families reported higher scores for depression and lower PRF than did the control group; only mothers showed lower parental alliance. Logistic regression highlighted the fact that several of these familial and parental factors contributed to the increased risk of belonging to the clinical group, specifically both mothers' and fathers' depressive symptoms and lower PRF. These data represent new findings with potentially meaningful clinical implications for both assessment and intervention., (Copyright © 2019 Mazzeschi, Buratta, Germani, Cavallina, Ghignoni, Margheriti and Pazzagli.)

Background/objective: In recent decades, the prevalence of childhood obesity has increased, with the major implications for public health. However, the factors that contribute to obesity in children are still poorly understood. The present study aimed to investigate the role of parental reflective functioning (PRF) in childhood obesity., Method: In a cross-sectional design, 120 sets of parents of 60 children ( n  = 30 with obesity, age range 6-11) were recruited by local paediatricians. Parents completed the Parental Reflective Functioning Questionnaire. Children's and parents' weight (assessed by BMI), as well as their socio-economic status (SES), were assessed to explore the contribution of PRF in the prediction of children's weight, controlling for parents' weight and SES., Results: t -test showed significant differences with medium effect sizes in BMI, SES and PRF between parents of children with and without obesity. The best model resulted from hierarchical multiple regression analyses and showed that mothers' PRF predicted children's BMI above and beyond the prediction by parents' BMI and SES., Conclusions: Low maternal PRF could be an important target for intervention strategies, highlighting the need to consider parental responses to children's emotions in the treatment of childhood obesity, particularly in parents with low SES and high BMI.

We compared each individual group member's attachment fit with the attachment of the other group members. Participants were 201 members of 20 therapy groups who sought treatment because they were overweight or obese. Group members completed the Attachment Style Questionnaire before commencing treatment and the Group Questionnaire at three points during group treatment. Multilevel polynomial regression followed by response surface analysis was used to examine how the attachment anxiety and avoidance of the individual group member and the aggregated attachment anxiety and avoidance of the group members were related to individual group members' perceptions of the positive bonding, positive working relationships, and negative relationships in the group. The findings suggested that attachment heterogeneity in group is related to the perception of members' stronger bonding relationships and lower negative relationships. In addition, positive bonding can be increased when a patient high in attachment avoidance is added to a group whose patients are low in attachment avoidance; however, group members reported weaker negative relationships when their attachment anxiety or avoidance became increasingly higher or lower than other group members' attachment anxiety or avoidance.

Pretend play has a central role for children's development and psychological well-being. However, there is a paucity of standardized and valid measures specifically devoted to assess the core domains involved in play activities in preschool and primary school children. The Affect in Play Scale-Preschool (4-5 years) and the Affect in Play Scale-Preschool Extended Version (6-10 years) are semi-structured parallel tools designed to explore child's cognitive and affective processes using a standardized play task. The current study administered this 5-min play task to 538 Italian children aged 4-10. The purposes were to compare play abilities in boys vs. girls and in preschool vs. primary school children, to correlate pretend play with divergent thinking and to evaluate the structural validity of the measure along the considered age span. No differences, excepting for Organization, were found between boys and girls, whereas school age children reported higher play abilities then the younger ones. External validity was assessed using correlational analysis with the divergent thinking task (the Alternate Uses Test) for preschoolers and primary school-aged children, in line with findings from Manova. Construct validity, assessed through the Confirmatory Factor Analysis, showed good fits for the two-factor model with cognitive and affective factor for both the Affect in Play Scale-Preschool and its Extended Version. A multi-group factor analysis suggested a partial invariance of the two-factor model across preschool (4-5 years old) and primary school-aged (6-10 years old) children. Results supported the use of the Affect in Play Scale-Preschool and its Extended Version as adequate measures to assess the interplay of cognitive and affective skills in preschool and school age children. The discussion highlights clinical and research implications linked to the possibility to have a unique play task able to assess child's affective and cognitive abilities throughout a quite wide life span (from 4 to 10 years old).

The transition to parenthood is widely considered a period of increased vulnerability often accompanied by stress. Abidin conceived parenting stress as referring to specific difficulties in adjusting to the parenting role. Most studies of psychological distress arising from the demands of parenting have investigated the impact of stress on the development of dysfunctional parent-child relationships and on adult and child psychopathology. Studies have largely focused on mothers' postnatal experience; less attention has been devoted to maternal prenatal characteristics associated with subsequent parental stress and studies of maternal prenatal predictors are few. Furthermore, no studies have examined that association exclusively with samples of first-time mothers. With an observational prospective study design with two time periods, the aim of this study was to investigate the role of mothers' attachment style, maternal prenatal attachment to the fetus and dyadic adjustment during pregnancy (7th months of gestation) and their potential unique contribution to parenting stress 3 months after childbirth in a sample of nulliparous women. Results showed significant correlations between antenatal measures. Maternal attachment style (especially relationship anxiety) was negatively correlated with prenatal attachment and with dyadic adjustment; positive correlations resulted between prenatal attachment and dyadic adjustment. Each of the investigated variables was also good predictor of parenting stress 3 months after childbirth. Findings suggested how these dimensions could be considered as risk factors in the transition to motherhood and in the very beginning of the emergence of the caregiving system, especially with first-time mothers.

Attention-Deficit/Hyperactivity Disorder (ADHD) is a disorder characterized by a chronic, pervasive, and developmentally inappropriate levels of impulsivity and in attention. It is associated with adverse academic and social functions and stress to families. Studies provide evidence that family variables are correlated with this disorder and that parenting styles play an important role in its complexity. However, a thorough investigation of the impact of parental affective and relational aspects on the ADHD child's areas of functioning is still needed. In designing future research on ADHD, we suggest to investigate parenting characteristics to a greater extent by adopting an attachment perspective with a focus on parental reflective functioning as it pertains to the child's ADHD clinical condition.

Interstitial lung disease, nephrotic syndrome and junctional epidermolysis bullosa is an autosomal recessive multiorgan disorder caused by mutations in the gene for the integrin α3 subunit (ITGA3). The full spectrum of manifestations and genotype-phenotype correlations is still poorly characterized. Here, we uncovered the disease-causing role and the molecular mechanisms underlying a homozygous ITGA3 mutation leading to the single amino acid substitution, p.R463W. The patient suffered from respiratory distress and episodes of cyanosis with onset in the first week of life and had a nephrotic syndrome. Although there was no clinical evidence for cutaneous fragility, the analysis of a skin sample and of skin epithelial cells enabled the direct assessment of the authentic mutant protein. We show that the mutation altered the conformation of the extracellular β-propeller domain of the integrin α3 subunit preventing correct processing of N-linked oligosaccharides, heterodimerization with β1 integrin and maturation through cleavage into heavy and light chains in the Golgi. Confocal microscopy demonstrated that the mutant protein accumulated intracellularly, but it was not present in focal adhesions or on the cell membrane as shown by flow cytometry. These findings highlight that single amino acid changes in the integrin α3 subunit may crucially alter the structure and complex processing of this integrin, completely preventing its functionality. The present report also underscores that ITGA3 mutations may account for atypical cases solely with early onset respiratory and renal involvement., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The study aimed to explore the differences in the role of specific personal and interpersonal risk factors in predicting fear of childbirth (FOC) and to examine whether FOC predicts postnatal maternal adaptation in nulliparous and parous women. A prospective correlational design with two time periods (pre- and postnatal) was carried out with 158 women, 85 nulliparous and 73 parous. Women at Week 32 of gestation completed a demographic questionnaire, the Wijma Delivery Expectancy Questionnaire (K.Wijma, B. Wijma, & M. Zar, 1998), the State-Trait Anxiety Inventory (C.D. Spielberger, R.L. Gorsuch, & R.E. Lushene, 1970), the Symptom Checklist-90-Revised (L.R. Derogatis, 1983), the Dyadic Adjustment Scale (G.B. Spanier, 1976), and the Reciprocal Attachment Questionnaire (M. West, A. Sheldon, & L. Reiffer, 1987). Three months after delivery, the women completed the Edinburgh Postnatal Depression Scale (J.L. Cox, J.M. Holden, & R. Sagovsky, 1987) and the Parenting Stress Index-Short Form (R. Abidin, 1986). Pearson's correlations and a series of multiple regressions were conducted. The results indicated that in the prenatal period, higher state anxiety, β = .35, p < .001, lower dyadic adjustment, β = -.26, p = .03, and higher insecurity in attachment relationships, β = .39, p < .001, predicted FOC in first-time mothers only. In the postnatal period, FOC predicted postnatal maternal risk for depression, β = .39, p = .02, and parenting stress, β = .42, p = .02, for nulliparous women only. The specific antecedents and consequences of FOC in nulliparous and parous women should be taken into consideration when developing specific interventions., (© 2014 Michigan Association for Infant Mental Health.)

The Circle of Security Parenting (COS-P) is an early attachment based intervention that can be used with groups, dyads, and individuals. Created in the USA and now used in many countries, COS-P is a visually based approach that demonstrates its central principles through videos of parent/child interactions. The core purpose of the COS-P is to provide an opportunity for caregivers to reflect on their child's needs and on the challenges each parent faces in meeting those needs. Even though there is a wide range of clinical settings in which child/parent attachment is an important component of assessment there is limited empirical data on when and how attachment based interventions are appropriate for specific clinical profiles and contexts. The aim of this paper is to present a clinical application of COS-P in order to explore and reflect on some specific therapeutic tasks where it works and on some clinical indicators and contexts appropriate for its application. A single case study of a father, "M." (43 years old) in conflict for the custody of his 5 years old daughter is reported. The Adult Attachment Projective Picture System (AAP), the Parenting Stress Index, the Strengths and Difficulties Questionnaire, and the Parental Alliance Measure, were administered pre- and post-intervention. The clinical significance analysis method revealed that numerous changes occurred in the father. The AAP showed improvements in the level of agency of self. M. made gains in his capacity to use internal resources and to increase his agency of self. M. was classified as recovered in his perception of the child's functioning and as improved in his parenting stress and parenting alliance with the mother. Considerations on specific contexts and clinical indicators for the application of COS-P are proposed.

Within the research area on the determinants of childhood obesity, a relatively new approach is the use of attachment theory to explore the mechanisms underlying children's obesity risk, especially considered as emotion regulation strategies in parent-child relationship. Few are the empirical researches that have addressed this issue. The empirical investigations have used self-report measures to assess adult attachment. In attachment studies, the use of interview methods and/or performance-based instruments is advised to evaluate the entire range of possible adult attachment patterns and comprehensively explain the emotional strategies, correlates, and consequences of individual differences in attachment system functioning. The aim of this study was to explore the extent to which both parents' attachment patterns serve as self-regulative mechanisms related to childhood overweight/obesity by the Adult Attachment Projective Picture System (AAP) in a sample of 44 mothers and fathers of children referred for obesity. Insecure attachment was found as a risk factor both for mothers and fathers. Also unresolved/disorganization was found to play a significant role in childhood obesity. The role of father's attachment was explored and findings suggested considering it in etiology and treatment of childhood obesity.

A time-lagged design was used to examine how the perceptions of alliance to the group as a whole by the other group members at an earlier point in the group were related to an individual group member's perceptions of alliance to the group as a whole at a later point in the group. We also examined how treatment outcome moderated this relationship. Seventy-three patients diagnosed as overweight or obese participating in 10 short-term therapy groups provided data for analyses. Group members completed measures of cohesion to the group and alliance to the group as a whole at the third, sixth, and last session of 12-session groups as well as pre- and posttest ratings on Obesity-Related Well-Being and the Outcome Questionnaire-45. As hypothesized, earlier ratings of alliance to the group as a whole by the other group members were related to later ratings of alliance to the group as a whole by the group member. Also as hypothesized, when group members had a better outcome, there was a significant positive relationship between perceptions of alliance to the group as a whole by the other group members at an earlier point in the group and an individual group member's perceptions of alliance to the group as a whole at a later point in time. When members had a worse outcome, there was no relationship between perceptions of alliance to the group as a whole by the other group members at an earlier point in the group and an individual group member's perceptions of alliance to the group as a whole at a later point in the group., ((PsycINFO Database Record (c) 2014 APA, all rights reserved).)

It is clinically relevant to understand whether it is safe to recommend to trained overweight/obese people long-distance treks and whether these experiences could have a negative psychological impact or become even dangerous exposing the trekkers to the risk of clinically silent myocardial damage. To answer these questions we have performed a quantitative/qualitative study comparing the changes in mood profiles, personal views, body composition, and plasma troponin levels of 40 overweight/obese subjects with those of 36 healthy normal weight subjects after the participation in a trek of 388 km from the Adriatic to the Tyrrhenian seas trek: the "Step by step…Italy's coast to coast". The results of this study demonstrate that long-distance treks are a safe activity for trained overweight/obese people which should be recommended because they improve mood, health status, and the relationship of participants with themselves and with the regular practice of exercise with effects similar to those obtained by healthy normal weight subjects.

The present paper describes the Epode Umbria Region Obesity Prevention Study (EUROBIS) and aims to implement the C.U.R.I.A.MO. model through the EPODE methodology. The main goal of the EUROBIS is to change the pendency of slope of the actual trend towards the increase in the yearly rates of childhood overweight and obesity in Umbria and to improve healthy lifestyles of children and their parents. The project is the first EPODE program to be performed in Italy. The aims of the Italian EUROBIS study are: (1) a community-based intervention program (CBP) carrying out activities in all primary schools of the Umbria Region and family settings as first step, to reverse the current obesity trend on a long-term basis, and (2) a clinical care program for childhood and adolescent by C.U.R.I.A.MO. model. C.U.R.I.A.MO. model is a multidisciplinary approach to improve three key aspects of healthy lifestyles: nutrition, exercise, and psychological aspects with the strategy of a family-based approach. The community-based intervention and clinical trial provide an innovative valuable model to address the childhood obesity prevention and treatment in Italy.

Objective: The aim of the present study was to investigate the maternal and paternal specific contributions on the associations between family socioeconomic status, parental anthropometric factors, parental alliance, and family functioning. These were assessed separately for mothers and fathers, by means of a case-control study with families of Italian youths with and without overweight/obesity., Methods: Ninety families with children aged 11 to 16 years (mean = 13.27 years; SD = 1.5) participated in the study. Half of the families included children with overweight/obesity (n = 45). The body mass indices (BMIs) of youths and parents were measured and the former transformed in BMI z-scores. The parents completed the Parenting Alliance Measure and the Family Assessment Measure Version III, General Scale., Results: Higher levels of dysfunction in parental alliance and family functioning of the mothers and fathers of the overweight/obese group were found. Socioeconomic status did not contribute significantly to the prediction of overweight and obesity in youth. Both maternal and paternal BMIs were positively associated with youth overweight/obesity. The degrees of parental alliance perceived by both mothers (odds ratio [OR], 81; 95% confidence interval [CI], 0.72-0.90) and fathers (OR, 89; 95% CI, 0.81-0.98) predicted child's weight status. The perception of poor parental and familial functioning by both parents contributed to the prediction of overweight and obesity in youth (mother: OR, 1.06; 95% CI, 1.02-1.10; father: OR, 1.06; 95% CI, 1.01-1.11)., Conclusion: The results support a strong effect of parental and family functioning on a youth's overweight/obesity also from the father's perspective. The importance of considering the father's perspective is discussed.

Bowlby (1973) stated that the attachment relationship between a child and parents might help the child learn many skills important for survival, with a lifelong effect. However, there is a scarcity of research into attachment and intelligence in adulthood. This study examines the relationship between attachment status and cognitive skills in an Italian non-clinical sample of 101 young adults (M age = 22.6 yr.; SD = 1.8). The Adult Attachment Projective Picture Stimuli and the Wechsler Adult Intelligence Scale-Revised were administered to participants and four-way attachment classifications were analyzed regarding participants' IQ. Statistically significant differences were found for Full Scale IQ and Verbal IQ, with Dismissing participants scoring higher than Unresolved participants. Similar results were obtained for two-way attachment patterns with Resolved participants scoring higher than Unresolved. Implications regarding different attachment patterns and the concept of trauma are discussed.

The current study investigates the effects of a multidisciplinary lifestyle intervention for obesity on Health Related Quality of Life (HRQoL). In this study, 92 adults with obesity were weighed and completed the Short Form Health Survey (SF-36) and the Center for Epidemiologic Studies Depression Scale (CES-D) at the baseline and at the end of a 3-month intervention. Repeated measures analysis of variance (ANOVA), a series of hierarchical regressions, and the statistical mediation analysis of Baron and Kenny were conducted. (a) Over 3 months, changes in weight and the mental and physical scales of the SF-36 and depression were all significant. (b) The results indicate a significant mediation of changes in depression scores for the association between weight loss and enhancement on the General Health scale of the SF-36. Improvements to HRQoL from a multidisciplinary lifestyle intervention seem to affect both the physical and mental components of the SF-36 score and to reduce depression, contributing to enhanced self-perceptions of well-being.

Context: There is scarce knowledge of the interaction between depression/health-related quality of life (HRQOL) and lifestyle intervention in obesity., Objective: The aim of the study was to establish whether baseline mood status or HRQOL affects attendance to educational or exercise sessions and whether attendance to these two components of the intervention affects mood and/or HRQOL in obesity., Design: A total of 282 overweight/obese subjects (body mass index, 33.4 ± 5.9 kg/m(2); 103 males, 179 females; age, 53.8 ± 13.0 yr, mean ± sd) were consecutively enrolled in a multidisciplinary lifestyle intervention program. During the intensive phase of the intervention (3 months) patients were invited to attend eight educational sessions and 26 exercise group sessions., Results: Poor adherence to exercise sessions is predicted by baseline depressive mood (P =0.006) and by low levels of HRQOL (domains of Vitality, Physical Role Functioning, Social Functioning, Mental Composite, Physical Composite Scores) (P < 0.05). Attendance to the educational sessions is associated with beneficial effects of the lifestyle intervention on depressive symptoms (P < 0.013) and on several mental domains of HRQOL (P < 0.041); attendance to the exercise sessions predicted the beneficial effects on perceived general health (P < 0.021) and body mass index (P < 0.011). Attendance to both educational and exercise components is associated (P < 0.05) with the reductions in waist circumference, fat mass, and blood pressure observed after the intensive phase of the intervention., Conclusions: Measurement of depressive mood and HRQOL before lifestyle intervention allows identification of patients at increased risk of attrition with exercise and educational sessions. Both the exercise and the educational sessions are essential for gaining the full spectrum of psychological and clinical benefits from multidisciplinary lifestyle intervention in obesity.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is the defective protein of the ichthyosiform condition Netherton syndrome (NS). Strongly expressed in the most differentiated epidermal layers, LEKTI is a serine protease inhibitor synthesized as three different high-molecular-weight precursors, which are rapidly processed into shorter fragments and secreted extracellularly. LEKTI polypeptides interact with several proteases to regulate skin barrier homeostasis as well as inflammatory and/or immunoallergic responses. Here, by combining antibody mapping, N-terminal sequencing, and site-specific mutagenesis, we defined the amino-acid sequence of most of the LEKTI polypeptides physiologically generated in human epidermis. We also identified three processing intermediates not described so far. Hence, a proteolytic cascade model for LEKTI activation is proposed. We then pinpointed the most effective fragments against the desquamation-related kallikreins (KLKs) and we proved that LEKTI is involved in stratum corneum shedding as some of its polypeptides inhibit the KLK-mediated proteolysis of desmoglein-1. Finally, we quantified the individual LEKTI fragments in the uppermost epidermis, showing that the ratios between LEKTI polypeptides and active KLK5 are compatible with a fine-tuned inhibition. These findings are relevant both to the understanding of skin homeostasis regulation and to the design of novel therapeutic strategies for NS.

Purpose: To illustrate how a thorough neurophysiological evaluation allows early diagnosis and prognosis of traumatic brachial plexus injuries., Methods: As examples, we report on the case of two patients with acute traumatic brachial plexus injuries for whom a comprehensive neurophysiological evaluation allowed early diagnosis and prognosis., Results: Neurophysiological findings were consistent with complete proximal (root) lesion in one patient and a severe neuroapraxic block between Erb point and axilla in the other one., Conclusions: The two reported acute cases of traumatic brachial plexus injuries demonstrated the high diagnostic and prognostic value of the neurophysiological tests when fully utilized.

Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling. In contrast, mutation to the negatively charged glutamic acid disrupted SH2 domain binding without reducing EphB2 kinase activity. By using a panel of EphB2 mutants, we found that kinase activity is required for the changes in cell-matrix and cell - cell adhesion, cytoskeletal organization, and activation of mitogen-activated protein (MAP) kinases elicited by EphB2 in transiently transfected cells. Instead, the two juxtamembrane SH2 domain binding sites were dispensable for these effects. These results suggest that phosphorylation of tyrosines 605 and 611 is critical for EphB2-mediated cellular responses because it regulates EphB2 kinase activity.

An important early event in the differentiation of skeletal muscle cells is exit from the cell cycle, after which full expression of the muscle phenotype occurs. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, expresses a number of muscle-specific proteins, including MyoD; however, these cells fail to arrest or differentiate when cultured in differentiation medium (DM). To determine the basis for the failure of RMS cells to differentiate or arrest, we studied the molecular response of the embryonal RMS cell line, RD, to culture in DM. Under these conditions, the retinoblastoma protein (RB) was primarily in the hyperphosphorylated state. This is in contrast to myoblasts cultured in DM, in which the hypophosphorylated form of RB is exclusively present. Measurements of the expression and activities of cyclin-dependent kinases (cdks) cdk2 and cdk4 indicated that RD cells maintained higher levels than do myoblasts, and the activity and abundance of these proteins did not significantly decrease upon culture in DM in RD cells, as they did in myoblasts. Similarly, elevated expression of cyclins D1, E, and A was observed in RD cells. Interestingly, cdk inhibitors are expressed in RD cells, with p16ink4 expression markedly elevated relative to myoblasts. Ectopic expression of p21cip1, p16ink4, or p27kip1 caused a growth arrest of RD cells but not detectable expression of a myogenic marker. Furthermore, a constitutively active RB protein could also inhibit the growth of RD cells without inducing myogenic differentiation. Taken together, these data suggest that the elevated levels of cdk2 and/or cdk4 observed in RD cells contribute to the inability of RD cells to growth arrest when cultured in DM but that these activities alone are not responsible for the failure of RD cells to differentiate.

This phase I-II study aimed to determine the maximum tolerated dose (MTD) of paclitaxel (Taxol), infused over 3 hours, when combined with a fixed dose (90 mg/m2) of epirubicin. Other aims were to investigate the combination's plasma pharmacokinetics, toxicity, and activity in 50 patients with previously untreated metastatic breast cancer, as well as its ability to mobilize peripheral blood stem cells (PBSCs). The initial dose of paclitaxel, 135 mg/m2, was increased by 20 mg/m2 in subsequent cohorts of six patients until dose-limiting toxicity (DLT) occurred. The DLT of the combination was febrile neutropenia in two of eight patients who received paclitaxel at 225 mg/m2. The concentration of epirubicinol, the major metabolite of epirubicin, decreased from 47.3 +/- 9.4 ng/mL at 175 mg/m2 of paclitaxel to 37.9 +/- 7.5 ng/mL at the 225-mg/m2 dose. The most relevant toxicity was grade 4 neutropenia (61% of all the courses). Cardiac toxicity included three patients (6%) developing congestive heart failure responsive to therapy. Among 49 evaluable patients, 41 responses (84%) were observed (95% confidence interval [CI], 70% to 92%) and 9 (19%) of these were complete. In 21 patients, we evaluated the mobilization of PBSCs after this regimen plus a colony-stimulating factor. The median number of CD34+ cells was 61.7/microL (range, 6.8 to 201/microL), and a median of 6.3 x 10(6)/kg of CD34+ cells have been harvested with a single leukapheresis.

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.

We studied the level of the cytosolic phosphotyrosine protein phosphatase (PTPase) (originally termed low-M(r) acid phosphatase) in normal NIH/3T3 and in v-erbB-transformed fibroblasts. The level of the enzyme, assayed by ELISA, was inversely related to cell proliferation, normally growing cells had less enzyme than their contact-inhibited counterparts and v-erbB transformants had less enzyme than normal NIH/3T3. In order to overexpress the enzyme and study its effects in normal and transformed cells, we transfected a synthetic gene coding for the PTPase in control NIH/3T3 and v-erbB transformants. The overexpressed enzyme was recognized by antibodies raised against the native enzyme and, in cells overexpressing the PTPase, we observed a marked dephosphorylation of tyrosyl residues of cellular proteins. Cell proliferation, in both normal and v-erbB transformants overexpressing the PTPase, was measured. We observed that PTPase overexpression was accompanied by significantly reduced thymidine incorporation in both cell types, either serum-starved or serum-stimulated. The ability of transformed v-erbB cells to grow in soft agar was also markedly decreased by overexpression of the enzyme. Taken together, our results indicate that overexpression of PTPase might interfere with mitogenic signalling pathways in both normal and transformed cells, and propose a role for PTPase in the control of cell proliferation.