Nonphosphorylated peptide ligands for the Grb2 Src homology 2 domain

Critical intracellular signals in normal and malignant cells are transmitted by the adaptor protein Grb2 by means of its Src homology 2 (SH2) domain, which binds to phosphotyrosyl (pTyr) residues generated by the activation of tyrosine kinases. To understand this important control point and to design inhibitors, previous investigations have focused on the molecular mechanisms by which the Grb2 SH2 domain selectively binds pTyr containing peptides. In the current study, we demonstrate that the Grb2 SH2 domain can also bind in a pTyr independent manner. Using phage display, an 11-amino acid cyclic peptide, G1, has been identified that binds to the Grb2 SH2 domain but not the src SH2 domain. Synthetic G1 peptide blocks Grb2 SH2 domain association (IC50 10-25 microM) with a 9-amino acid pTyr-containing peptide derived from the SHC protein (pTyr317). These data and amino acid substitution analysis indicate that G1 interacts in the phosphopeptide binding site. G1 peptide requires a YXN sequence similar to that found in natural pTyr-containing ligands, and phosphorylation of the tyrosine increases G1 inhibitory activity. G1 also requires an internal disulfide bond to maintain the active binding conformation. Since the G1 peptide does not contain pTyr, it defines a new type of SH2 domain binding motif that may advance the design of Grb2 antagonists.     

SPECT scatter modelling in non-uniform attenuating objects

When young children appear to recognize that someone else is engaging in make-believe play, do they infer what the pretender is thinking? Are they aware that the pretender is thinking about a pretend scenario yet knows what the real situation is? Preschoolers ages 3-5 (N = 45) viewed scenes from the Barney & Friends television series depicting either make-believe or realistic actions. Children were questioned concerning the presence of pretense and the thoughts and beliefs of the TV characters. The children were also presented with false belief and appearance/reality theory of mind tasks. Children who identified when TV characters were engaging in pretend play did not necessarily infer the pretenders' thoughts and beliefs. Inferring pretenders' thoughts was related to performance on false belief and appearance/reality tasks, but simply recognizing pretense was not. These data support the view that children initially learn to recognize pretense from contextual cues and are able to infer pretenders' beliefs only with further development of metarepresentational ability.     

Fractionated high-dose-rate brachytherapy in primary carcinoma of the nasopharynx

PURPOSE: A growing body of data suggests that local control in nasopharyngeal cancer (NPC) is related to the radiation dose administered. We conducted a single-institution study of high-dose radiotherapy (RT), which incorporated high-dose-rate (HDR) brachytherapy (BT). These results were analyzed together with data obtained from controls who did not receive BT. PATIENTS AND METHODS: The BT group comprised 42 consecutive patients of whom 29 patients were staged according to the tumor, node, metastasis system as T1 through 3, 13 patients were T4, and 34 patients were N+ disease. BT was administered on an outpatient basis by means of a specially designed flexible nasopharyngeal applicator, and the dose distributions were optimized. Treatment for T1 through 3 tumors comprised 60 Gy of external-beam radiotherapy (ERT) followed by six fractions of 3 Gy BT (two fractions per day). Patients with parapharyngeal tumor extension and/or T4 tumors received 70 Gy ERT and four fractions of 3 Gy BT. The no-BT group consisted of all patients treated from 1965 to 1991 (n = 109), of whom 82 patients had stages T1 through 3, 27 patients had T4, and 80 patients had N+ disease. Multivariate Cox proportional hazards analyses were performed by using the end points time to local failure (TTLF), time to distant failure (TTDF), disease-free survival (DFS), cause-specific survival (CSS), and the prognostic factors age, tumor stage, node stage, and grade. Because the overall treatment time varied substantially in the no-BT group, the dependence of local failure (LF) on the physical dose as well as the biologic effective dose (BED) corrected for the overall treatment time (OTT) (BEDcor10) was studied. RESULTS: The BT group had a superior 3-year local relapse-free rate (86% v 60%; univariate analysis, P = .004). Multivariate analysis showed hazards ratios for BT versus no-BT of 0.24 for TTLF (P = .003), 0.35 for TTDF (P = .038), 0.31 for DFS (P < .001), and 0.44 for CSS (P = .01). The best prognostic group consisted of patients with T1 through 3, N0 through 2b tumors treated with BT who attained a 5-year TTLF of 94% and CSS of 91%. In contrast, the worst prognostic group, i.e., 5-year TTLF of 47% and CSS of 24%, was composed of patients with T4 and/or N2c through 3 tumors who did not receive BT. CONCLUSION: High doses of radiation (73 to 95 Gy) can be administered to patients with NPC with minimal morbidity by means of optimized HDR-BT. The use of a BT boost proved to be of significant benefit, particularly in patients with T1 through 3, N0 through 2b disease. The steep dose-effect relationship seen for the physical dose and the BEDcor10 indicates that the results are dose related. The analysis has identified a poor prognostic group in whom treatment intensification with chemotherapy (CHT) is indicated.     

Characterization of the genes encoding D-amino acid transaminase and glutamate racemase, two D-glutamate biosynthetic enzymes of Bacillus sphaericus ATCC 10208

In Bacillus sphaericus and other Bacillus spp., D-amino acid transaminase has been considered solely responsible for biosynthesis of D-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding D-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28. 8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the D-amino acid transaminase of the thermophilic Bacillus sp. strain YM1.     

Distribution and excretion of radiolabelled tert-butylhydroquinone in Fischer 344 rats

Uniformly 14C-ring-labelled tert-butylhydroquinone (TBHQ) was diluted with non-radioactive TBHQ and administered orally (for excretion studies) to Fischer 344 rats. An average of 72.9% and 10.6% of the administered radioactivity was recovered in the urine and faeces, respectively, of male rats, and 77.3% and 8.2% in the urine and faeces, respectively, of female rats in 4 days. No significant sex-related differences were found in either excretion, tissue distribution or urinary metabolites of TBHQ-derived radiolabel. For distribution studies, intraperitoneal doses were administered to female rats, and tissue levels of radiolabel were determined at various times after dosing. The parent compound quickly disappeared from tissue in vivo. The highest concentrations of radiolabel were found in the liver and kidneys. The urinary metabolites consisted of conjugated TBHQ and unidentified polar substance(s).     

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.     

Recognition specificity of individual EH domains of mammals and yeast

The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.     

Quality adjusted survival analysis with repeated quality of life measures

One way of examining trade-offs between quantity and quality of life (QOL) is to combine them into a single measure such as quality-adjusted life year (QALY). If censoring occurs, then estimation presents some difficulties. One approach, known as Q-TWiST, is to define a series of health states, use a 'partitioned' survival analysis to calculate the average time in each state, and then weight each state according to its quality of life to calculate QALYs. Such health-state models, however, are unhelpful when the transitions between health states are unclear or if they do not adequately reflect variations in quality of life. We therefore examine an alternative analysis to be used when repeated measures of quality of life are available from individual patients in a clinical trial. The method proceeds by separating quality of life and survival, that is, dQALY/dt = S(t)Q(t), where S(t) is the survival curve, estimated from the standard Kaplan-Meier method, and Q(t) is the quality of life function, derived from individual repeated measures of quality of life. We derive single health-state (QALY) and multiple health-state (Q-TWiST) models and illustrate the approach by comparing different durations of adjuvant chemotherapy for breast cancer.     

A case controlled comparison of open and laparoscopic splenectomy in children

BACKGROUND: This case controlled study compares the efficacy, safety, and cost of laparoscopic splenectomy (LS) and open splenectomy (OS) for hematologic disorders in children. METHODS: The records of 82 consecutive children and adolescents undergoing splenectomy for hematologic disorders between August 1994 and September 1997 were reviewed retrospectively. RESULTS: Fifty patients underwent LS by a lateral approach and 32 underwent OS through a left subcostal incision. Mean age was 7.76 years for LS and 6.9 years for OS. Patient weights were similar: (LS, mean 30.5 kg; OS, mean 27.6 kg). Hematologic indications included hereditary spherocytosis in 43 children (LS 26, OS 17), sickle cell anemia with sequestration in 13 (LS 7, OS 6), immune thrombocytopenic purpura in 14 (LS 8, OS 6), and 12 with other disorders (LS 9, OS 3). Concomitant cholecystectomy was performed in 10 of 50 LS and 6 of 32 OS cases. Accessory spleens were identified in 8 of 32 (25%) OS and 9 of 50 (18%) LS cases (P = .578). No LS procedures required conversion to OS. The mean estimated blood loss was 54.4 mL for LS and 49.0 mL for OS (P = .233). LS required a longer operative time (115 vs 83 minutes, P = .002), less need for postoperative intravenous narcotic (51% vs 100%, P < .0001), lower total narcotic doses (0.239 vs 0.480 mg/kg morphine, P = .006), shorter length of hospital stay (1.4 +/- 0.97 vs 2.5 +/- 1.43 days, P = .0001), and lower average total hospital charges ($5713 vs $6564) than OS. There were no deaths or major complications in either group. CONCLUSIONS: Laparoscopic splenectomy is a safe and effective procedure in children with hematologic disorders resulting in longer operative times, less narcotic administration, shorter length of stay, and lower total hospital charge.