Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase

Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn24, Ala25, and Asp26 at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg2+ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active site. It is concluded that the metal ion binding site found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ greatly stabilizes the enzyme hexamer but has little effect on catalysis.     

Developmentally regulated expression of persyn, a member of the synuclein family, in skin

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.     

Segmental antigen challenge increases fibronectin in bronchoalveolar lavage fluid

Fibronectin may contribute to asthma pathogenesis by recruitment and activation of inflammatory cells, and by promotion of subepithelial fibrosis. Fibronectin is produced by several types of airway cells, including epithelial cells, fibroblasts, and alveolar macrophages. To test the hypothesis that antigen-induced airway inflammation is associated with increased local generation of fibronectin, segmental bronchoprovocation (SBP) with antigen and saline was performed in 17 atopic patients. Bronchoalveolar lavage (BAL) was performed at 5 min and 48 h after segmental challenge with saline or antigen. Fibronectin concentrations in BAL fluid, measured by enzyme-linked immunosorbent assay (ELISA), increased more than 5-fold 48 h after antigen challenge (65 [47 to 110] versus 407 [240 to 697] ng/ml, median and 25 to 75% interquartiles, p < 0.05). Fibronectin concentrations 48 h after antigen challenge correlated with histamine concentrations 5 min after antigen challenge and numbers of eosinophils, neutrophils, macrophages, and total cells in BAL fluid 48 h after antigen challenge. BAL was more enriched in fibronectin 48 h after challenge than would be predicted solely from increased permeability of plasma proteins. Western blot analysis showed that fibronectin in BAL fluid was largely intact and contained the extra domain-A (ED-A) splice variant of cellular fibronectin, indicative of local production. We conclude that antigen challenge in atopic subjects causes increased production of fibronectin by airway cells and speculate that this response may contribute to airway remodeling in allergic inflammation.     

Insulin therapy could prevent salt-induced high blood pressure in diabetes mellitus

The effectiveness of insulin replacement therapy in the prevention of salt-induced hypertension in diabetes mellitus was examined using Alloxan diabetic rats. Early daily (eight units/day) treatment with insulin prevented the development of high blood pressure after six weeks of high-salt feeding. The mean arterial pressure (MAP) for the early insulin-treated and salt-fed group (DET-SF) was 123.37 +/- 6.37 mmHg which was close to the value for normal (control) rats 128.17 +/- 4.84 mmHg, but significantly (p < 0.001) less than that of the untreated diabetic salt-fed group (DSF) which was 164.58 +/- 8.33 mmHg. The nondiabetic salt-fed (NDSF) group had MAP of 150.27 +/- 4.24 mmHg. Late commencement of insulin therapy did not significantly affect the sensitivity of the diabetic rats to high-salt diet. The results suggest that early commencement of insulin therapy could prevent the development of high blood pressure in diabetic rats.     

Mechanism of the radiation-protective effect of indralin

Output from the interpositus nucleus can inhibit the inferior olive, probably via the GABA-ergic nucleo-olivary pathway. It has been suggested that the function of this inhibition might be to regulate synaptic plasticity resulting from parallel fibre/climbing fibre interaction in cerebellar Purkinje cells, by providing negative feedback information to the olive. Thus, when a learned response, generated by the interpositus nucleus, reaches a sufficient amplitude, the olive would be inhibited and further learning blocked. This suggestion was tested in a classical conditioning paradigm. Decerebrate ferrets were trained using electrical skin stimulation of the forelimb as the conditioned stimulus (CS) and periorbital stimulation as the unconditioned stimulus (US). Climbing fibre responses evoked in Purkinje cells by the US were recorded as surface field potentials in the part of the c3 zone controlling eyeblink. It was found that the CS did not inhibit the olive at the beginning of training, but when conditioned responses were large, the olive was inhibited by the CS in some animals. After a number of unpaired CS presentations, which caused extinction of the conditioned response, the inhibition disappeared. The size of individual conditioned responses correlated negatively with the size of the climbing fibre responses evoked by the US. Climbing fibre responses evoked by direct stimulation of the olive were also inhibited. It was concluded that cerebellar output during performance of a conditioned response inhibits the inferior olive. The results thus support the hypothesis of a cerebellar locus of conditioning and are consistent with the proposed role of cerebello-olivary inhibition.     

Effect of alpha-tocopherol on lipid peroxidation and total antioxidant status in spontaneously hypertensive rats

The aim of this study was to determine the effects of alpha-tocopherol on lipid peroxidation and total antioxidant status of spontaneously hypertensive rats (SHR), comparing them with normal Wistar-Kyoto (WKY) rats. SHR were divided into three groups and treated with different doses of alpha-tocopherol (alpha1, 17 mg/kg diet; alpha2, 34 mg/kg diet; and alpha3, 170 mg/kg diet). Normal WKY and untreated SHR were used as normal (N) and hypertensive control (HC). Blood pressures were recorded every 10 days for 3 months. At the end of the trial, animals were killed and measurement of plasma total antioxidant status, plasma superoxide dismutase (SOD) activity, and lipid peroxide levels in plasma and blood vessels was carried out following well-established methods. From our study it was found that lipid peroxides in thoracic aorta (N, 0.47 +/- 0.17; H, 0.96 +/- 0.37; P < .0001) and plasma (N, 0.06 +/- 0.01; H, 0.13 +/- 0.01) were significantly higher in hypertensives than in normal rats. SOD activity was significantly lower in hypertensive than normal rats (N, 172.93 +/- 46.91; H, 110.08 +/- 14.38; P < .005). Total antioxidant status was significantly higher in normal than hypertensive rats (N, 0.88 +/- 0.05; H, 0.83 +/- 0.02; P < .05). After the antioxidant trial, it was found that in the treated groups rise of blood pressure was prevented significantly (P < .001) and lipid peroxides in blood vessels were significantly reduced more than in the controls (P < .001). For plasma lipid peroxide it was only significant for groups alpha2 (P < .001) and alpha3 (P < .05). Although all three treated groups showed improved total antioxidant status, only groups alpha2 (0.87 +/- 0.04, P < .005) and alpha3 (1.20 +/- 0.18, P < .001) were statistically significant. All the three groups showed significant increases in their SOD activity (P < .001). Correlation studies showed that total antioxidant status and SOD were significantly negatively correlated with blood pressure in normal rats (P = .007; P = .008). Lipid peroxides in both blood vessel and plasma showed a positive correlation. In the treated groups, lipid peroxides in blood vessels maintained a significant positive correlation with blood pressure in all groups (alpha1, P = .021; alpha2, P = .019; alpha3, P = .002), whereas for plasma lipid peroxides the correlation was in groups alpha1 (P = .005) and alpha2 (P = .009). For SOD activity, significant negative correlations were found with blood pressure in the alpha2 (P = .017) and alpha3 (P = .025) groups. Total antioxidant status maintained a significant negative correlation with blood pressure in all three groups (alpha1, P = .012; alpha2, P = .044; alpha3, P = .014). In conclusion it was found that supplement of alpha-tocopherol may prevent development of increased blood pressure, reduce lipid peroxides in plasma and blood vessels, and enhance the total antioxidant status, including SOD activity.     

Studies of small GTPase Rab7 association with endosomes of cells expressing normal and mutant forms of epidermal growth factor receptors

A study was made of an association of small GTPase Rab7, commonly considered as a marker of late endosomes, with endosomal compartments of cells expressing EGF receptor with different ability to be sorted for degradative pathway. It was found that in cells HER14, expressing normal EGF receptor, Rab7 was associated with both early and late endosomes and the extent of association correlated with the number of EGF-receptor complexes in the specific endosomal fraction. Cels with a receptor, lacking major sites of autophosphorylation by deletion of 126 C-terminal residues (CD126), demonstrated a low efficiency of EGF-receptor sorting to late endosomes and decreased association dynamics with Rab7. Interaction of Rab7 with endosomes of cells expressing kinase negative receptors (K721) was found to be minimal. At the same time, in cells Cd126 and K721 with a low sorting efficiency Rab7 was mainly associated with early endosomes. These data favor Rab7 involvement in mediating early-to-late endosomal transition.     

Local recurrence of breast cancer after cytological evaluation of lumpectomy margins

Successful breast conservation therapy with optimal cosmesis requires adequate tumor excision and negative tumor margins. Therefore, more sensitive techniques are being developed to identify lumpectomy margins intraoperatively with greater accuracy. Unidentified microscopic disease is seemingly responsible for a local recurrence rate of up to 25 per cent 3 to 5 years after lumpectomy and radiotherapy for breast cancer patients. As a result, Moffitt Cancer Center has routinely used an intraoperative touch preparation cytology (TPC) protocol to evaluate the entire resected surface of all lumpectomies. In addition, resection margins were also evaluated by gross examination and by standard histology. In rare instances frozen sections were used to evaluate tumor margins. In this study 701 consecutive lumpectomy specimens were evaluated by TPC during the period of 9 years with a mean follow-up of 3.5 years. Local cancer recurrence was 2.7 per cent (mean recurrence, 2.53 years), in women whose lumpectomy margins were evaluated by TPC. Of interest, a local recurrence rate of 14.6 per cent was observed in patients who had referral lumpectomies evaluated by conventional histopathology. This study suggests that accurate margin assessment with TPC plays an important role in the control of local recurrence after breast conservation therapy. Therefore, we conclude the routine use of intraoperative TPC provides rapid, reliable, topographically accurate identification of residual microscopic disease at lumpectomy margins.     

The immediate and late allergic response to segmental bronchopulmonary provocation in asthma

The response to antigen is an important factor in the development of airway inflammation. Segmental bronchoprovocation (SBP) with antigen and subsequent bronchoalveolar lavage (BAL) have provided valuable insight into the mechanisms of allergic inflammation. To determine the features of allergic airway response in asthma, 19 subjects with mild asthma underwent antigen SBP in a dose-dependent manner. The amount of antigen used in SBP was 0 (saline), and 1, 5, or 20% of the antigen dose required to drop the FEV1 by 20% (APD20). BAL was done at 5 min and 48 h after SBP. BAL histamine levels increased modestly 5 min after antigen SBP. At 48 h, there was a marked increase in eosinophils and IL-5 concentration even in airway segments where the release of histamine was small. Moreover, eosinophils correlated with IL-5 levels at 48 h (r = 0.63; p < 0.001), but not with BAL histamine concentrations at 5 min. GM-CSF levels did not increase after antigen SBP and did not correlate with eosinophils. These observations indicate that asthmatic subjects can develop a dose-dependent response to antigen SBP that is characterized by a modest increase in histamine immediately after antigen exposure, and marked eosinophilia, which appears proportionately greater than the histamine response and relatively greater than what is seen in allergic nonasthmatic subjects. This feature might be important to the eventual development of airway inflammation in asthma.