Requirement of Src kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating factor

Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.     

alpha-Melanocyte stimulating hormone inhibits tumour necrosis factor-alpha stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines

alpha-Melanocyte stimulating hormone (alpha-MSH) was found significantly to reduce tumour necrosis factor-alpha (TNF-alpha) stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in normal adult cutaneous melanocytes. The maximum inhibitory response to alpha-MSH was obtained at around 10(-10) mol/L alpha-MSH when cells were coincubated with alpha-MSH and TNF-alpha for 24 h. alpha-MSH had little or no effect on basal ICAM-1 expression in melanocytes and the effects of alpha-MSH could be mimicked with 3-isobutyl-1-methylxanthine (IBMX). Preliminary data in three human melanoma cell lines also showed alpha-MSH and forskolin to be effective in significantly reducing TNF-alpha stimulated ICAM-1 expression over 24 h. The extent of the inhibition varied from cell line to cell line and was greatest in those cells with the highest number of alpha-MSH receptors. These data suggest that alpha-MSH has the ability to oppose the action of the pro-inflammatory cytokine TNF-alpha on melanocytes and melanoma cells.     

125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes

Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2], both nonradioactive [I-Tyr1]biphalin and radioactive [125I-Tyr1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I-Tyr1]biphalin to the delta and mu opioid receptors are not independent. [125I-Tyr1]Biphalin binds to delta receptors as shown in NG108-15 cell membranes. Nevertheless, [125I]biphalin binding to delta receptors in rat brain membranes was hardly evident and mu receptor binding predominated or at least was much more readily detectable in this preparation.     

A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

Relationship between patient reports of urinary incontinence symptoms and quality of life measures

The aim of the present study was to assess the effect of normal growth and development on the Peer Assessment Rating index between 12 and 22 years of age. The sample consisted of 49 nonorthodontic subjects (24 male and 25 females) from the Nijmegen Growth Study. The mean age at the first observation was 12.2 +/- 0.7 years and at the second observation 22.1 +/- 0.6 years. The dental casts at these ages were scored with the Peer Assessment Rating index. The influence of the Angle classification and the treatment need on changes over time in the Peer Assessment Rating score were evaluated. The mean changes in the PAR scores over time between cases that have more than 30% improvement to those that have less than 30% improvement were calculated. The results indicate that there were no significant differences between the mean Peer Assessment Rating score at 12 years of age (12.20 +/- 0.91) and at 22 years of age (12.45 +/- 1.28), but there were relevant differences in individual cases. The changes were irrespective of the Angle classification or the treatment need. Changes over time in the weighted Peer Assessment Rating score were mainly correlated to changes in the anterior crossbite and the overjet. This correlation may be influenced, however, by the applied weighting factor for those occlusal traits.     

Isolation of translactone-containing triterpenes with thrombin inhibitory activities from the leaves of Lantana camara

Methanolic extracts prepared from the leaves of Lantana camara have been found to inhibit human thrombin. An assay, in which thrombin activity is measured as a function of clot formation from fibrinogen, was used to guide the fractionation and purification of five principal active constituents (1-5), which were all characterized as 5,5-trans-fused cyclic lactone-containing euphane triterpenes.     

Rack-induced metal binding vs. flexibility: Met121His azurin crystal structures at different pH

The rack-induced bonding mechanism of metals to proteins is a useful concept for explaining the generation of metal sites in electron transfer proteins, such as the blue copper proteins, that are designed for rapid electron transfer. The trigonal pyramidal structure imposed by the protein with three strong equatorial ligands (one Cys and two His) provides a favorable geometry for both cuprous and cupric oxidation states. However, the crystal structures of the Met121His mutant of azurin from Alcaligenes denitrificans at pH 6.5 (1.89- and 1.91-A resolutions) and pH 3.5 (2.45-A resolution) show that the preformed metal binding cavity in the protein is more flexible than expected. At high pH (6.5), the Cu site retains the same three equatorial ligands as in the wild-type azurin and adds His121 as a fourth strong ligand, creating a tetrahedral copper site geometry with a green color referred to as 1.5 type. In the low pH (3.5) structure, the protonation of His121 causes a conformational change in residues 117-123, moving His121 away from the copper. The empty coordination site is occupied by an oxygen atom of a nitrate molecule of the buffer solution. This axial ligand is coordinated less strongly, generating a distorted tetrahedral copper geometry with a blue color and spectroscopic properties of a type-1 site. These crystal structures demonstrate that blue copper proteins are flexible enough to permit a range of movement of the Cu atom along the axial direction of the trigonal pyramid.     

Hypohydration effects on skeletal muscle performance and metabolism: a 31P-MRS study

The purpose of this study was to determine whether hypohydration reduces skeletal muscle endurance and whether increased H+ and Pi might contribute to performance degradation. Ten physically active volunteers (age 21-40 yr) performed supine single-leg, knee-extension exercise to exhaustion in a 1.5-T whole body magnetic resonance spectroscopy (MRS) system when euhydrated and when hypohydrated (4% body wt). 31P spectra were collected at a rate of one per second at rest, exercise, and recovery, and were grouped and averaged to represent 10-s intervals. The desired hydration level was achieved by having the subjects perform 2-3 h of exercise in a warm room (40 degrees C dry bulb, 20% relative humidity) with or without fluid replacement 3-8 h before the experiment. Time to fatigue was reduced (P < 0.05) by 15% when the subjects were hypohydrated [213 +/- 12 vs. 251 +/- 15 (SE) s]. Muscle strength was generally not affected by hypohydration. Muscle pH and Pi/beta-ATP ratio were similar during exercise and at exhaustion, regardless of hydration state. The time constants for phosphocreatine recovery were also similar between trials. In summary, moderate hypohydration reduces muscle endurance, and neither H+ nor Pi concentration appears to be related to these reductions.     

The dental, craniofacial, and biochemical features of pyknodysostosis: a report of three new cases

1.Near-infrared (IR) spectroscopy is based on the relative transparency of skin, skull and brain to the light in the near-IR region (700-1100 nm) and on the oxygen-dependent tissue absorption changes of haemoglobin.2. We evaluated the most relevant factors (reproducibility, venous return, age and sex) that might affect reliability of near-IR spectroscopy to test CO2 cerebrovascular reactivity.3.Thirty-four healthy volunteers were enrolled in the study. The protocol consisted of a 3-min baseline, a 3-min hypercapnia (5% CO2 in air) and a 2-min recovery. Transcranial Doppler sonography measurements were simultaneously performed. The CO2 reactivity test was repeated on 27 subjects after 1 h to assess reproducibility. CO2 reactivity was also evaluated at different body positions (supine, 35 degrees Trendelenburg and 35 degrees reverse Trendelenburg), and over a gradual increase of the inspired CO2.4. Changes in near-IR spectroscopy and transcranial Doppler sonography parameters were significantly correlated with variations of end-tidal CO2 (P<0.005). A significant correlation between the reactivity indexes of near-IR spectroscopy parameters and flow velocity was also found (P<0.01). A high reproducibility was also found for deoxyhaemoglobin (rI=0.76), oxyhaemoglobin (rI=0.68) and flow velocity (rI=0.60) reactivity indexes. No significant differences between the reactivity indexes of different body positions were found (P>0.05). The reactivity index of oxyhaemoglobin and deoxyhaemoglobin decreased (P<0.05) and increased (P<0.01) with age respectively.5. We found that near-IR spectroscopy is a reliable and reproducible method for the evaluation of cerebrovascular reactivity and might be considered, after appropriate validation, for the assessment of patients with cerebrovascular disease.     

Management of incomplete spontaneous abortion with suction curettage in the pediatric emergency department

Medical records, angiograms and operative records of 28 patients with partial anomalous pulmonary venous connection (PAPVC) were reviewed. Twenty patients had one anomalous pulmonary vein (APV), and 8 had more than two APVs. Twenty-five patients (89%) had APVs originating from the right lung, 2 (7%) from the left lung and 1 (4%) from both lungs. In the 25 patients with APVs originating from the right lung, 9 had APVs draining into the superior vena cava (SVC), 13 into the right atrium (RA), 1 into the inferior vena cava (IVC) and 2 into both the SVC and RA. In the 2 patients with APVs originating from the left lung, 1 had APVs draining into the RA, and the other had APVs draining into the innominate vein. The patient with APVs originating from both lungs had connection to the IVC. Twenty-three patients (82%) had additional cardiovascular defects. Surgery was performed in 13 patients who had pulmonary/systemic flow ratios greater than 2.0. The patients have done well after surgery. In 7 patients, we were unable to accurately determine the number or sites of drainage of APVs prior to surgery. We conclude that selective pulmonary angiography is indispensable for the accurate diagnosis of PAPVC.