Osteopenic consequences of botulinum toxin injections in the masticatory muscles: a pilot study.

Patients with temporomandibular muscle and joint disorder (TMJD) increasingly seek and receive treatment for their pain with botulinum toxin (BoNTA; botulinum toxin A). Used intramuscularly in therapeutic doses, it produces localised paresis. Such paresis creates risk of reduced bone mineral density, or 'disuse osteopenia'. Animal studies have frequently used BoNTA as a model of paralysis to induce bone changes within short periods. Osteopenic effects can be enduring in animals but have yet to be studied in humans. This is the first study in humans to examine bone-related consequences of BoNTA injections in the masticatory muscles, comparing oral and maxillofacial radiologists' ratings of trabecular bone patterns in the condyles of patients with TMJD exposed to multiple masticatory muscle injection sessions with BoNTA to a sample of patients with TMJD unexposed to masticatory muscle injections with BoNTA. Cone-beam computed tomography (CBCT)-derived images of bilateral condyles were evaluated in seven patients with TMJD receiving 2+ recent BoNTA treatment sessions for facial pain and nine demographically matched patients with TMJD not receiving BoNTA treatment. Two oral and maxillofacial radiologists evaluated CBCT images for evidence of trabecular changes consistent with osteopenia. Both evaluators noted decreased density in all participants exposed to BoNTA and in none of the unexposed participants (P < 0.001). No other abnormalities associated with reduced loading were detected. These findings need replication in a larger sample and over a longer time period, to ensure safety of patients with TMJD receiving multiple BoNTA injections for their pain.

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.

Higher cortical deficits influence attentional processing in dementia with Lewy bodies, relative to patients with dementia of the Alzheimer's type and controls.

BACKGROUND: Attentional dysfunction is believed to be a prominent and distinguishing neuropsychological feature of dementia with Lewy bodies (DLB); yet, the specific nature of the attentional deficit and factors that can potentially influence attentional processing in DLB have not been fully defined. AIMS: To clarify the nature of the attentional deficit in early-stage DLB relative to patients with early-stage dementia of the Alzheimer's type (DAT) and elderly controls, and examine the effect of task complexity and type of cognitive load on attentional processing in DLB. METHODS: Attentional impairment and fluctuating attention were investigated in three groups of subjects--patients with clinical features of early probable DLB (n = 20), a group with early probable DAT (n = 19) and healthy elderly controls (n = 20)--using an experimental computerised reaction time paradigm. RESULTS: Patients with DLB showed greater attentional impairment and fluctuations in attention relative to patients with DAT and elderly controls. The attentional deficit was generalised in nature but increased in magnitude as greater demands were placed on attentional selectivity. Attentional deficits in DLB were most pronounced under task conditions that required more active recruitment of executive control and visuospatial cognitive processes. CONCLUSIONS: Attentional deficits in DLB are widespread and encompass all aspects of attentional function. Deficits in higher cortical function influence the degree of attentional impairment and fluctuating attention, suggesting that attentional processing in DLB is mediated by interacting cortical and subcortical mechanisms. These findings serve to clarify the nature of the attentional deficit in DLB and have potentially important ramifications for our understanding of the neurocognitive underpinnings of fluctuations.

Mass spectrometric and thermodynamic studies reveal the role of water molecules in complexes formed between SH2 domains and tyrosyl phosphopeptides.

BACKGROUND: SH2 domains have a fundamental role in signal transduction. These domains interact with proteins containing phosphorylated tyrosine residues and, in doing so, mediate the interactions of proteins involved in tyrosine kinase signalling. The issue of specificity in SH2 domain interactions is therefore of great interest in terms of understanding tyrosine kinase signal-transduction pathways and in the discovery of drugs to inhibit them. Water molecules are found at the interfaces of many complexes, however, to date little attention has been paid to their role in dictating specificity. RESULTS: Here we use a combination of nanoflow electrospray ionization mass spectrometry (ESI-MS), isothermal titration calorimetry and structural data to investigate the effect of water molecules in complexes formed between the SH2 domain of tyrosine kinase Src and tyrosyl phosphopeptides. Binding studies have been performed using a series of different peptides that were selected to allow changes in the water content at the complex interface and demonstrate changes in specificity. ESI-MS enables quantification of the number of water molecules that interact with a higher affinity than those generally found solvating the biomolecular complex. CONCLUSIONS: Comparing the interactions of different peptides, we show that an intricate network of water molecules have a key role in dictating specificity. The use of mass spectrometry to quantify tightly bound water molecules may prove of general use in structural biology, where an independent determination of the water molecules associated with a structure would be advantageous. Furthermore, the ability to assess whether given water molecules are important in high-affinity binding could make this method a precious tool in drug design.

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.

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.

Relaxin inhibits spontaneous and prostaglandin-driven myometrial activity in anaesthetized rats.

Porcine relaxin (250 guinea-pig units/mg) infused intravenously into anaesthetized rats at 20 micrograms/h reversibly abolished spontaneous intra-uterine pressure cycles yet left the myometrium responsive to oxytocin in doses of 4--8 mu. The inhibition was found to be primarily of the frequency, rather than of the amplitude, of pressure cycles. Relaxin (5 or 10 micrograms) was capable of completely suppressing uterine activity driven by prostaglandin F2 alpha infusion in oestrogen-treated ovariectomized rats. Whereas the beta-adrenergic blocker, propranolol, had no effect on relaxin-induced inhibition, phentolamine, an alpha-blocker, significantly delayed the relaxin effect. It is unlikely, however, that relaxin operates through an alpha-inhibitory receptor. The results show that relaxin acts primarily as a frequency modulator and is capable of antagonizing an exogenous myometrial stimulant.

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.

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.

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.

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.

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.

Inconsistent detection of PrP in extraneural tissues of cats with feline spongiform encephalopathy.

Feline spongiform encephalopathy (FSE), a transmissible spongiform encephalopathy or prion disease of cats, first reported in Great Britain in 1990, is believed to result from the consumption of food contaminated by the agent of bovine spongiform encephalopathy (BSE). The accumulation of PrP in non-neural tissues of cats diagnosed as suffering from FSE was investigated by immunohistochemistry. In the majority of the cats no disease-specific PrP was detected in lymphoid tissues. Small amounts of PrP were detected in the spleen of only two of 13 samples examined, in Peyer's patches of one of the two cases for which suitable material was available, but in the myenteric plexus of all four cats in which sections of intestine were examined. In addition PrP immunostaining was found in the kidney of all the cats with FSE whose kidneys were examined.

Bovine spongiform encephalopathy: investigation of phenotypic variation among passive surveillance cases.

Bovine spongiform encephalopathy (BSE) is a prion disease of domesticated cattle, first identified in Great Britain (GB) in 1986. The disease has been characterized by histopathological, immunohistochemical, biochemical and biological properties, which have shown a consistent disease phenotype among cases obtained by passive surveillance. With the advent of active surveillance in 2001, immunological tests for detection of the prion protein revealed some cases with different biochemical characteristics and, in certain instances, differences in pathology that have indicated variant phenotypes and the possibility of agent strain variation. This study examines a case set of 523 bovine brains derived from archived material identified through passive surveillance in GB. All cases conformed to the phenotype of classical BSE (BSE-C) by histopathological, immunohistochemical and biochemical approaches. The analyses consolidated an understanding of BSE-C and, by western blotting, confirmed differentiation from the known atypical BSE cases which exhibit higher or lower molecular masses than BSE-C (BSE-H and BSE-L respectively).

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.

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics.

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees

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.

Demonstration of some of the physiological properties of rat relaxin.

Relaxin was extracted from the ovaries of pregnant rats. Material possessing uterine relaxing activity in vitro was eluted in three peaks from Sephadex G.50 columns. The 'G3 peak' material eluting in a position comparable to that of porcine relaxin inhibited myometrial activity of rats in vivo, improved the rate of rise of pressure of intrauterine pressure cycles in vivo, and, when administered to rats following progesterone and oestrogen priming, increased the distensibility of the cervix in vitro. This material also stimulated inter-pubic ligament formation in the mouse.. This rat relaxin material therefore exhibits biological actions in the rat similar to those previously assigned to porcine relaxin.