Oncogenic activation of JAK3-STAT signaling confers clinical sensitivity to PRN371, a novel selective and potent JAK3 inhibitor, in natural killer/T-cell lymphoma.

Aberrant activation of the JAK3-STAT signaling pathway is a characteristic feature of many hematological malignancies. In particular, hyperactivity of this cascade has been observed in natural killer/T-cell lymphoma (NKTL) cases. Although the first-in-class JAK3 inhibitor tofacitinib blocks JAK3 activity in NKTL both in vitro and in vivo, its clinical utilization in cancer therapy has been limited by the pan-JAK inhibition activity. To improve the therapeutic efficacy of JAK3 inhibition in NKTL, we have developed a highly selective and durable JAK3 inhibitor PRN371 that potently inhibits JAK3 activity over the other JAK family members JAK1, JAK2, and TYK2. PRN371 effectively suppresses NKTL cell proliferation and induces apoptosis through abrogation of the JAK3-STAT signaling. Moreover, the activity of PRN371 has a more durable inhibition on JAK3 compared to tofacitinib in vitro, leading to significant tumor growth inhibition in a NKTL xenograft model harboring JAK3 activating mutation. These findings provide a novel therapeutic approach for the treatment of NKTL.

A retrospective study of 286 cases of neurological disorders of the cat.

Archive central nervous tissue from 286 cats with neurological disorders was reviewed for histological evidence of feline spongiform encephalopathy (FSE), which may have occurred before it was first recognized in 1990. The following six categories of disease were identified: congenital; degenerative; inflammatory; neoplastic; FSE; lesion-free. The largest category (inflammatory) contained 92 cats, of which 47 were considered to be consistent with infection by feline infectious peritonitis (FIP) virus. Six cats showed evidence of more than one disease process; thus, one cat with FIP also had toxocara infection of the lateral ventricles and five cats with FSE also showed perivascular cuffing suggestive of concurrent viral infection. In only two cases did the diagnosis on review differ significantly from the original interpretation. There was no evidence of FSE before the original case was recognized in April 1990.

Feline necrotising sialometaplasia: a report of two cases.

Unilateral swelling of submandibular salivary gland in two cats was diagnosed as necrotising sialometaplasia. Histological features that differentiate the disease from other salivary gland lesions, particularly neoplasia are: lobular necrosis of salivary tissue; squamous metaplasia conforming to duct and/or acinar outlines; preservation of salivary lobular morphology; and variable inflammation and granulation tissue.

Mutational investigation of the specificity determining region of the Src SH2 domain.

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.

Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.

The binding of tyrosine phosphorylated targets by SH2 domains is required for propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not well understood. In order to identify the attributes of pTyr required for high affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoserine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosphopeptide. The dephosphorylated peptide and the phosphoserine containing peptide both bind extremely weakly to the Src SH2 domain (DeltaGo (dephosphorylated)=-3.6 kcal/mol, DeltaGo (phosphoserine) >-3.7 kcal/mol); however, the DeltaGo value of pTyr binding is more favorable (-4.7 kcal/mol, or 50 % of the entire binding free energy of a high affinity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affinity binding. Alanine mutagenesis was also used to evaluate the energetic contribution to binding of ten residues located in the pTyr-binding site. Mutation of the strictly conserved Arg betaB5 resulted in a large increase in DeltaGo (DeltaDeltaGo=3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (DeltaDeltaGo<1.4 kcal/mol) reduction in affinity, indicating that Arg betaB5 is the single most important determinant of pTyr recognition. However, mutation of Cys betaC3, a residue unique to the Src SH2 domain, surprisingly increased affinity by eightfold (DeltaDeltaGo=-1.1 kcal/mol). Using a double mutant cycle analysis, it was revealed that residues of the pTyr-binding pocket are not coupled to the peptide residues C-terminal to the pTyr. In addition, comparison of each residue's DeltaDeltaGo value upon mutation with that residue's sequence conservation among SH2 domains revealed only a modest correlation between a residue's energetic contribution to pTyr recognition and its conservation throughout evolution. The results of this investigation highlight the importance of a single critical interaction, the buried ionic bond between the phosphate of the pTyr and Arg betaB5 of the SH2 domain, driving the binding of SH2 domains to tyrosine phosphorylated targets.

Physician participation in research surveys. A randomized study of inducements to return mailed research questionnaires.

The authors randomly selected 400 physicians from a population of 1,545 practicing physicians providing follow-up care to patients who received bone marrow or blood stem cell transplants at the Fred Hutchinson Cancer Research Center to determine interest in receiving Internet-based transplant information. In a two-factor completely randomized factorial design, the 400 physicians were assigned to receive mailed surveys with either no compensation or a $5 check and either no follow-up call or a follow-up call 3 weeks after mailing. Overall, 51.5% of the physicians returned the mailed surveys. Comparison of logit models showed that inclusion of a $5 check in the mailer significantly (p = .016) increased the probability of returning the surveys (57.5% vs. 45.5%). In contrast, the telephone follow-up had no overall effect. The authors concluded a modest financial reward can significantly improve physician response rates to research surveys but a telephone follow-up may be inefficient and even ineffective.

Calorimetric investigation of proton linkage by monitoring both the enthalpy and association constant of binding: application to the interaction of the Src SH2 domain with a high-affinity tyrosyl phosphopeptide.

The binding of Src homology 2 (SH2) domains to tyrosyl phosphopeptides depends on electrostatic interactions between the phosphotyrosine and its binding site. To probe the role of these interactions, we have used isothermal titration calorimetry to study the pH dependence of the binding of the SH2 domain of the Src kinase to a high-affinity tyrosyl phosphopeptide. Two independent approaches were employed. In a first series of experiments that focused on determining the peptide's association constant between pH 5.0 and 9.0, two ionizable groups were characterized. One group, with free and bound pKas of 6.2 and 4.4, respectively, could be identified as the phosphate in the phosphotyrosine while the other group, with free and bound pKas of 8.2 and 8.5, respectively, could be only tentatively assigned to a cysteine in the phosphotyrosine binding pocket. Further information on the linkage between peptide binding and protonation of the phosphotyrosine was obtained from a second series of experiments, which focused on determining the peptide binding enthalpy at low values of pH in several buffers with different ionization enthalpies. These data provided free and bound pKa values for the phosphotyrosine identical to those derived from the first series of experiments, and hence demonstrated for the first time that the two approaches provide identical information regarding proton linkage. In addition, the second series of experiments also determined the intrinsic enthalpy of binding of both the protonated and deprotonated phosphate forms of the peptide. These two sets of experiments provided a complete energetic profile of the linkage between phosphate ionization and peptide binding. From this profile, it was determined that the PO32- form of the peptide binds 2.3 kcal mol-1 more favorably than the PO3H1- form due entirely to a more favorable entropy of binding.

Probing the "two-pronged plug two-holed socket" model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study.

Src homology 2 (SH2) domains are protein modules that specifically bind to tyrosyl phosphorylated peptides on signaling proteins. X-ray crystallographic studies of the SH2 domain of the Src kinase have probed the mechanism of binding, leading to the "two-pronged plug two-holed socket" mechanism whereby binding is hypothesized to resemble a two-pronged plug (the peptide) inserting into a two-holed socket (the SH2 domain). This binding model predicts (1) a hydrophobic basis for high-affinity binding largely determined by the level of insertion of the third residue C-terminal to the phosphotyrosine in the peptide into a primarily hydrophobic pocket (the +3 binding pocket) of the SH2 domain, and (2) a binding mechanism involving no significant conformational changes in the SH2 domain. In this study, we have probed these predictions by using isothermal titration calorimetry to extract complete thermodynamic profiles (Delta G degrees, Delta H degrees, Delta S degrees, Delta Cp degrees) for the binding of the Src SH2 domain to two series of tyrosyl phosphopeptides. One series consisted of peptides that have been determined by X-ray crystallography to have different levels of insertion of the peptide's +3 position into the +3 binding pocket. The other series consisted of peptides with progressively smaller hydrophobic side chains (I, L, V, and A) at the +3 position. Consistent with a binding mechanism that does not involve substantial conformational changes, the Delta Cp degrees values for all peptides were small and, at least for the high-affinity interactions, similar to the Delta Cp degrees values predicted from surface area calculations. However, unexpectedly, this study reveals that high-affinity binding was only partially determined by the interactions between the +3 residue in the peptide and the +3 binding pocket. Furthermore, the Delta Cp degrees values for all peptides studied were similar, implying similar degrees of desolvation of the +3 binding pocket upon binding. These results indicate that the "two-pronged plug two-holed socket" model is an oversimplification of the Src SH2 domain binding mechanism.

CARE-PARTNER: a computerized knowledge-support system for stem-cell post-transplant long-term follow-up on the World-Wide-Web.

Evidence-based practice in medicine promotes the performance of medicine based upon proven and validated practice. The CARE-PARTNER system presented here is a computerized knowledge-support system for stem-cell post-transplant long-term follow-up (LTFU) care on the WWW, which means that it monitors the quality of the knowledge both of its own knowledge-base and of its users. Its aim is to support the evidence-based practice of the LTFU clinicians and of the home-town physicians who actually care for the transplanted patients. Currently, three fundamental characteristics of CARE-PARTNER are accountable for its knowledge-support function: the quality of its knowledge-base, its availability on the WWW, and its learning from experience capability. As a matter of fact, the integration of a case-based reasoner in the reasoning framework enables the system to introspectively study its results, and to learn from its successes and failures, thus confronting the quality of the guidelines and pathways it reuses to the reality and complexity of the clinical cases.

Dysuria associated with urethral caruncle in the dog.

Three cases of urethral caruncle were recognized in bitches with a history of chronic dysuria. Clinical and radiological examinations revealed the presence of inoperable lesions involving much of the urethra in all three cases. At post-mortem examination of Case 1, an oval swelling, 1.5 x 1.0 cm, was detected within the wall of the urethra close to the vagino-urethral orifice. In Case 2, firm, mottled yellow, white and red tissue formed a thickening between the urethra and vagina. In Case 3, a cylindrical cream mass, 8 cm long and 3 cm in diameter, surrounded the urethra and impinged on the wall of the vagina. Histologically, glandular structures lined by a single layer of epithelial cells and a mixed granulomatous inflammatory reaction were present in the wall of the urethra of all three cases.

Plasma concentrations of prolactin, progesterone, relaxin and oestradiol-17 beta in sows treated with progesterone, bromocriptine or indomethacin during late pregnancy.

Pregnant gilts (3/group) were given no treatment, 10 mg bromocriptine twice daily by mouth, from Day 111 of pregnancy to 1 day post partum, 25 mg progesterone s.c. at 6-h intervals from Days 111 to 116 inclusive or 400 mg indomethacin by mouth at 6-h intervals from Day 111 to 116 inclusive. Before spontaneous delivery maternal plasma prolactin and relaxin concentrations started to rise almost simultaneously between 58 and 47 h before the first piglet and both hormones reached peak values when the plasma progesterone concentration had started to decline rapidly (approximately 21-23 h). Suppression of prolactin levels by bromocriptine prevented the onset of lactation completely but had no obvious influence on changes of the other hormone concentrations and the course of parturition. Progesterone treatment delayed the onset of expulsion of the piglets but did not delay the simultaneous increase in prolactin and relaxin concentrations. These changes in hormone levels were prevented by indomethacin treatment but occurred essentially unchanged when the treatment was ended. The results support the concept that parturition in the pig is preceded by a biphasic increase of plasma prostaglandin levels.

The timing and possible mechanisms of cervical opening in the oestrous rat.

The time that the retention of luminal fluid begins and its release from the uterus in pro-oestrous rats was determined precisely using a dye injection technique. Oestradiol was found to cause dye retention in ovariectomized rats when provided continuously from an implant of 5 mg. Injections of 0.5 microgram/d X 4 were ineffective. Administration or progesterone to rats bearing implants of oestrogen interfered with the ability of the uterus to retain dye. Treatment with relaxin for 28 h prevented retention of eye in pro-oestrous rats whereas treatment at the time of dye injection was without effect. Mechanical stimulation of the cervix did not cause loss of injected dye. It is suggested that the mechanisms of opening of the cervix at coitus and at the end of oestrous may differ.

Inhibition of oxytocin-or prostaglandin F2alpha-driven myometrial activity by relaxin in the rat is oestrogen-dependent.

Relaxin in doses of 5 microgram i.v. completed but reversibly abolishes "spontaneous' myometrial activity in anaesthetized ovariectomized rats. Similar levels of myometrial activity, evoked in oestrogen-treated rats (which normally have quiescent uteri) by infusions of oxytocin or prostaglandin F2alpha (PGF2alpha), were also reduced to complete quiescence by relaxin in small doses. However when spontaneous myometrial activity in untreated ovariectomized rats was slightly stimulated by oxytocin the uterus became completely refractory to the inhibitory effects of relaxin even at doses of 50 microgram. Relaxin was also ineffective in reducing myometrial activity in similar rats during intra-arterial infusion of PGF2alpha. It is suggested that the ability of relaxin to inhibit uterine smooth muscle during exogenous stimulation is oestrogendependent.

Rat myometrial activity in vivo: effects of oestradiol-17 beta and progesterone in relation to the concentrations of cytoplasmic progesterone receptors.

The amplitude, frequency and rate of rise of intra-uterine pressure cycles in rats (postpartum, ovariectomized) were unaffected by treatment with progesterone. Amplitude was also unaffected by a combination of treatments with progesterone and oestradiol-17 beta, which was adequate to ensure the survival of 84% of foetuses in ovariectomized pregnant rats. The failure of progesterone to influence myometerial activity could not be attributed to a lack of "true" progesterone receptors since these were present in the myometria of the test animals in concentrations exceeding those of oestrous animals. Evidence was obtained which suggested that a high-affinity binding protein, different from the "true" receptor may predominate in the myometrium of the pregnant rat. Oestradiol-17 beta in single or repeated doses of from 0.25 to 5 microgram, however, was found to reduce the frequency of pressure cycles but to increase significantly their rate of rise of pressure. There was a latency of 6--8 h in these effects of oestradiol. The possibility that inhibition of the myometrium by oestrogen may play a part in the preparation for parturition is discussed.