IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3.

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.

Suppression of lipopolysaccharide-stimulated cytokine/chemokine production in skin cells by sandalwood oils and purified α-santalol and ß-santalol.

Medicinally, sandalwood oil (SO) has been attributed with antiinflammatory properties; however, mechanism(s) for this activity have not been elucidated. To examine how SOs affect inflammation, cytokine antibody arrays and enzyme-linked immunosorbent assays were used to assess changes in production of cytokines and chemokines by co-cultured human dermal fibroblasts and neo-epidermal keratinocytes exposed to lipopolysaccharides and SOs from Western Australian and East Indian sandalwood trees or to the primary SO components, α-santalol and ß-santalol. Lipopolysaccharides stimulated the release of 26 cytokines and chemokines, 20 of which were substantially suppressed by simultaneous exposure to either of the two sandalwood essential oils and to ibuprofen. The increased activity of East Indian SO correlated with increased santalol concentrations. Purified α-santalol and ß-santalol equivalently suppressed production of five indicator cytokines/chemokines at concentrations proportional to the santalol concentrations of the oils. Purified α-santalol and ß-santalol also suppressed lipopolysaccharide-induced production of the arachidonic acid metabolites, prostaglandin E2, and thromboxane B2, by the skin cell co-cultures. The ability of SOs to mimic ibuprofen non-steroidal antiinflammatory drugs that act by inhibiting cyclooxygenases suggests a possible mechanism for the observed antiinflammatory properties of topically applied SOs and provides a rationale for use in products requiring antiinflammatory effects.

Evidence for alternate splicing within the mRNA transcript encoding the DNA damage response kinase ATR.

Proper cellular response to genotoxic insult often requires the activity of one or more members of a family of high-molecular weight protein kinases referred to as phosphatidylinositol-3 kinase (PIK)-like proteins. While catalytic activity is an indispensable part of PIK-like protein function, little is currently known about factors that control their activity and/or functions. This deficiency stems, in large part, from our lack of knowledge concerning functionally significant subdomains within the large non-catalytic domain of these proteins. We have determined that the transcript encoding the PIK-like protein ATR undergoes alternate splicing within the region of the mRNA encoding its non-catalytic domain. This conclusion is based on the sequencing of a human expressed sequence tag clone encoding a portion of the ATR cDNA, and is supported by the results of reverse transcriptase-polymerase chain reaction (RT-PCR) assays conducted on total and polyA+ RNA, as well as sequencing of cloned RT-PCR products. Cloning and sequencing of a segment of human genomic DNA indicated that this event arises from splicing of a single 192 bp exon within the ATR gene. Analysis of several human tissues indicated that alternate ATR transcripts are differentially expressed, suggesting that this region of the ATR protein may be of functional importance.

The ZNF217 gene amplified in breast cancers promotes immortalization of human mammary epithelial cells.

The functional consequences of overexpression of a candidate oncogene on chromosome 20q13.2, ZNF217, were examined by transducing the gene into finite life span human mammary epithelial cells (HMECs). In four independent experiments, ZNF217-transduced cultures gave rise to immortalized cells. HMECs that overcame senescence initially exhibited heterogeneous growth and continued telomere erosion, followed by increasing telomerase activity, stabilization of telomere length, and resistance to transforming growth factor beta growth inhibition. The incremental changes in telomerase activity and growth that occurred in ZNF217-transduced cultures after they overcame senescence were similar to the conversion pattern we have described previously in rare HMEC lines immortalized after exposure to a chemical carcinogen. Aberrant expression of ZNF217 may be selected for during breast cancer progression because it allows breast cells to overcome senescence and attain immortality.

Dementia with Lewy bodies treated with rivastigmine: effects on cognition, neuropsychiatric symptoms, and sleep.

Dementia with Lewy bodies (DLB) is a common cause of the dementia syndrome. Symptomatic treatment of the fluctuating cognition, visual hallucinations, and sleep disturbance that characterize this condition is challenging; neuroleptics are relatively contraindicated. We describe eight patients fulfilling the consensus diagnostic criteria for probable DLB who were treated with rivastigmine. Clinical features rated were: cognition by the Modified Mini-Mental State Examination (3MS); and behavioral and psychiatric symptoms by the Neuropsychiatric Inventory (NPI). Additional information was obtained from family and nursing reports. Seven patients showed resolution or improvement in cognition and neuropsychiatric symptoms as demonstrated by improvement in their 3MS and NPI scores. They also became more independent in mobility and activities of daily living, and the majority returned to live in their own home. Of the seven patients with sleep disruption, six improved. One case had no improvement in his symptomatology and the rivastigmine was stopped. Outcomes in this case series suggest that rivastigmine is well tolerated in clinical practice.

A built-in arginine finger triggers the self-stimulatory GTPase-activating activity of rho family GTPases.

Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound state and the inactive GDP-bound state. Rho family members containing an arginine residue at position 186 in the C-terminal polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction, resulting in significantly enhanced intrinsic GTPase activities. This arginine residue functions effectively as an "arginine finger" in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding interactions of Rho family proteins. The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes. Introduction of the arginine 186 mutation into S. cerevisiae CDC42 led to phenotypes consistent with down-regulated CDC42 function. Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative regulation.

Development in release testing of topical dosage forms: use of the Enhancer Cell with automated sampling.

The aim of this study was to evaluate an automated method using the Enhancer Cell and compare the release of the corticosteroid triamcinolone acetonide (TA) from commercial semisolid formulations. The method used a modified USP Apparatus 2 using the Enhancer Cell in 200 ml capacity flasks instead of the standard 900 ml flasks. The additional equipment included an adapter plate to position the flasks in the center, a cover to reduce the receptor phase evaporation and smaller sized (1/4 in.) shaft and collets. All products were evaluated prior to their expiration date. Effects of system variables such as the temperature and composition of the receptor medium, stirring speed, and the choice of membrane on the drug release were evaluated. Statistical analysis was carried out using SAS Ver. 6.07 and the slopes and intercepts (of the cumulative release/unit area versus square root of time plots) were compared. TA release was a linear function of the square root of time (P < or = 0.0001), in accordance with Higuchi's model (r2 > or = 0.9 in most cases). Temperature (32 and 37 degrees C) did not affect the drug release (P > 0.32) but a significantly higher release rate was observed (P < or = 0.0001) at 50 degrees C. Stirring speed (50, 100, 200 rpm) (P > 0.26) and receptor media composition (38 and 76% ethanol) (P > 0.68) did not significantly alter the release rates. Membrane selection (regenerated cellulose, polyethylene, and rat skin) was found to be a significant variable (P < or = 0.004). This study demonstrates the use of the Enhancer Cell as an automated quality control tool in the in vitro release testing procedure for semisolid drug formulations.

Analysis of the mechanisms of action of the Saccharomyces cerevisiae dominant lethal cdc42G12V and dominant negative cdc42D118A mutations.

The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42(D118A) mutant and the lethal, gain of function cdc42(G12V) mutant were examined. Cdc42(D118A,C188S)p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the two-hybrid system, which correlated with the temperature dependence of the cdc42(D118A) phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42(D118A) action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42(D118A, C188S)p-Cdc24p interactions and could suppress the cdc42(D118A) mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42(G12V) mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1Delta and cla4Delta cells but not ste20Delta cells, suggesting that the mechanism of cdc42(G12V) action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42(G12V) were also identified that displayed a dominant negative phenotype at 16 degrees C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.

Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.

Alagille syndrome is an autosomal dominant disorder characterized by abnormal development of liver, heart, skeleton, eye, face and, less frequently, kidney. Analyses of many patients with cytogenetic deletions or rearrangements have mapped the gene to chromosome 20p12, although deletions are found in a relatively small proportion of patients (< 7%). We have mapped the human Jagged1 gene (JAG1), encoding a ligand for the developmentally important Notch transmembrane receptor, to the Alagille syndrome critical region within 20p12. The Notch intercellular signalling pathway has been shown to mediate cell fate decisions during development in invertebrates and vertebrates. We demonstrate four distinct coding mutations in JAG1 from four Alagille syndrome families, providing evidence that it is the causal gene for Alagille syndrome. All four mutations lie within conserved regions of the gene and cause translational frameshifts, resulting in gross alterations of the protein product Patients with cytogenetically detectable deletions including JAG1 have Alagille syndrome, supporting the hypothesis that haploinsufficiency for this gene is one of the mechanisms causing the Alagille syndrome phenotype.

Effect of varying drug loading on particle size distribution and drug release kinetics of verapamil hydrochloride microspheres prepared with cellulose esters.

Microspheres containing two different drug loadings of a calcium channel blocker, verapamil hydrochloride, were prepared with three different cellulose esters namely cellulose acetate (CA), cellulose acetate propionate (CAP) and cellulose acetate butyrate (CAB) of approximately similar molecular weights using the emulsion-solvent evaporation method. Increasing the drug loading from 33.3 to 50% w/w increased the geometric mean diameter of the microspheres as well as the T50% values, i.e. time required to release 50% of the drug from microspheres prepared with all the three cellulose esters. Drug release from the microspheres was affected by the nature of polymer. Mathematical modelling of drug release data by fitting the data to various equations revealed that the data did not fit the conventional Higuchi's and Baker-Lonsdale's models for drug release from spherical matrices. Instead, the data fitted the log-probability and the Weibull models quite well.

Prognostic value of c-erbB-2 and epidermal growth factor receptor in stage A1 (T1a) prostatic adenocarcinoma.

OBJECTIVE: To determine whether the presence or absence of the oncoproteins epidermal growth factor receptor (EGFR) and c-erbB-2 could predict tumour behaviour. PATIENTS AND METHODS: Tissue from 45 stage A1 (T1a) prostatic adenocarcinomas from patients with a mean age of 65 years were immunostained for EGFR (12E) and c-erbB-2 (NCL-CB11). Their expression in the tumour and surrounding benign hyperplastic epithelium was correlated with each other and with survival. RESULTS: Forty percent (18 of 45) and 36% (16 of 45) of patients respectively were EGFR and c-erbB-2 positive in the tumour. Expression of these tyrosine kinase oncogenes was not confined to the tumour and the surrounding hyperplastic prostate was also positive for EGFR in 76% (34/45) of patients and for c-erbB-2 in 16% (11 of 45). EGFR and c-erbB-2 expression was weakly associated in both benign and malignant epithelium. Statistical analysis of survival showed that tumour c-erbB-2 expression was associated with a significantly worse prognosis (exact two tailed P = 0.0316), whereas no significant association was observed between EGFR expression and survival (P = 0.737). CONCLUSION: As c-erbB-2 expression increases the rate of dying by 4.2 times, recording its expression by these tumours may be useful in selecting patients who would benefit from treatment in stage A1 (T1a) disease.

The measurement of the lateralization of narrow bands of noise using an acoustic pointing paradigm: the effect of sound-pressure level.

The effects of varying interaural time delay (ITD) and interaural intensity difference (IID) were measured in normal-hearing subjects as a function of eleven frequencies and at sound-pressure levels (SPL) from 60 to 90 dB SPL and at 25-dB sensation level. Using an "acoustic" pointing paradigm, the IID of a 500-Hz narrow-band (100 Hz) noise (the "pointer") was varied by the subject to coincide with that of a "target" ITD stimulus. ITDs of 0, +/- 200, and +/- 400 microseconds were obtained through total waveform delays of narrow-band noise (NBN), including envelope and fine structure. The results of this experiment confirm the traditional view of binaural hearing for like stimuli: There is little perceived displacement away from 0 IID at frequencies of 1250 Hz and above. In the low frequencies, subjects required IIDs greater than the expected 10 dB to perceive a fully lateralized image, and they varied in the maximum value of IID that they required, regardless of frequency. Our subjects did not always perceive the intracranial locations of ITD targets symmetrically: When the signal was delayed to one ear, the resultant matching IID was often different in magnitude than for the same ITD target delayed to the opposite ear for the identical frequency. The results of two subjects suggested that people with asymmetric normal hearing have adapted to their asymmetry for lateralization tasks: The subjects were found to lateralize toward the ear with the greater SPL stimulus, regardless of the ear to which the signal was delayed, when signals of equal SL were presented, and toward the leading ear when signals of equal SPL were presented (unequal SL). Increasing the presentation levels above 60 dB SPL had an effect on the perception of high-frequency ITD targets: As the intensity level increased, the slopes of the IID versus ITD functions increased indicating better discrimination of ITD. This study is in agreement with other studies in providing strong evidence of individual differences in lateralization experiments. These individual differences might be attributable to differential sensitivity to ambiguous time stimulus cues, differential task sensitivity, age effects, threshold asymmetries, or criterion variability.

Evaluation of Preflo modified starches as new direct compression excipients. I. Tabletting characteristics.

This investigation evaluated some new (Preflo) and existing commercially available (Starch 1500, Star Tab) modified starches as direct compression excipients. Preflo corn starches (CH-10, CH-20, CH-30) and Preflo potato starches (P-250, PI-10, PJ-20) were evaluated and compared with respect to their pharmaceutical properties such as particle size, density, flowability, friability, and compression properties. Preflo starches showed a high bulk density and good flowability. Preflo corn starches and Star Tab formed harder tablets than Preflo potato starches and Starch 1500. Data from the Athy-Heckel plots indicated that the Preflo starches are soft materials and, unlike Starch 1500, undergo plastic deformation. Tablets containing acetaminophen were also compressed with the starches and disintegration and dissolution studies were conducted. Starch 1500 tablets disintegrated in 3.5 min, whereas none of the Preflo starch tablets disintegrated in 30 min. While complete acetaminophen release occurred in 25 min from Starch 1500 tablets, the drug dissolution time from Preflo starch tablets varied from 4 to 12 hr, indicating a potential use for some of these starches in solid oral modified-release dosage forms.

p53 and c-myc expression in stage A1 prostatic adenocarcinoma: useful prognostic determinants?

Forty-five stage A1 prostatic adenocarcinomas from patients with a mean age of 65 years were examined for p53 and c-myc expression to determine whether the presence or absence of these proteins could predict tumor behavior. Thirteen (6 of 45) and seventy-three percent (33 of 45) of cases were respectively p53 and c-myc positive. p53 expression was confirmed to the tumor cells, whereas c-myc immunoreactivity was present in both malignant and surrounding hyperplastic prostate. Statistical analysis showed that although p53 and c-myc expression were positively correlated, expression of neither nuclear protein was associated with a significantly worse survival (p53: p = 0.0791 exact two-tailed; c-myc: p = 0.738 exact two-tailed). These results suggest that while both p53 and c-myc may play a role in prostatic carcinogenesis, neither appears to identify patients who may benefit from treatment in stage A disease.

Low prevalence of human papillomavirus types 16 and 18 in cervical adenocarcinoma in situ, invasive adenocarcinoma, and glandular dysplasia by polymerase chain reaction.

Using the polymerase chain reaction on paraffin blocks for human papillomavirus (HPV) types 16 and 18, a positive result was seen in three of 20 cases of invasive adenocarcinoma, 15 of 36 cases of adenocarcinoma in-situ, and one of five cases of glandular dysplasia of the uterine cervix. Types 16 and 18 were found with equal frequency. In adenocarcinoma in situ there was no age difference between HPV-positive and or HPV-negative cases. Concomitant squamous dysplasia was slightly more frequent in HPV-positive adenocarcinoma in-situ. HPV positivity rates are lower than most of those previously reported. Possible reasons for this are discussed.

Eye muscle prosthesis.

We inserted a silicone rubber elastic band along the course of a paralyzed lateral rectus and of a paralyzed superior oblique to restore alignment and to provide a spring against which the antagonist could pull. The lateral rectus band has been in place for 7 years. It provides alignment and a field of single binocular vision of 20 degrees. The superior oblique band has been in place for 17 months. It provides alignment and single vision over 30 degrees from the primary position except for a restriction in upgaze-adduction to 25 degrees (Brown syndrome) and in downgaze-adduction to 20 degrees. Such engineered elastic bands are a useful addition to current surgical techniques for management of cases of paralysis and restriction.

Polymerase chain reaction and allele-specific oligonucleotides in paternity testing of the deceased.

The assignment of paternity when the alleged father has died is now possible by use of a variety of allele-specific oligonucleotides after amplification of genomic DNA by the polymerase chain reaction (PCR). Issues relating to the inheritance of estates may be decided on fact rather than allegation. PCR-based genotyping of DQ alpha haplotypes from paraffin-embedded tissue of the deceased was used to prove non-paternity in the case reported here. Because the child was female, it was also possible to confirm the exclusion by using a second polymorphic site located in the factor VIII gene on the X chromosome.

Length-tension recording system for strabismus surgery.

To meet the need for both scientific information and a clinical means for measurement of the mechanical parameters of the most difficult individual strabismus cases we present a technique for directly measuring and plotting the length-tension characteristics of the tissues supporting the eye. Semiconductor strain gauges mounted on the shanks of a custom machined eye forceps and an ultrasonic method of making continuous duction measurements of the eye have proved feasible. When the forceps are interfaced with a dedicated microcomputer, the system provides a permanent, quantitative, length-tension record displayed in real-time. The instrumented length-tension forceps system has provided a noninvasive means for quickly and simply assessing the mechanical underlying determinants of strabismus pathology in the office, the laboratory or in the operating room, and can aid in the planning and immediate intraoperative alteration of strabismus surgery. Under operator coordination, measurements can be made which precisely define the mechanical load which the eye muscles must move. The resulting objectively determined tissue stiffness asymmetries and muscle restrictions limiting ocular motion indicate the purely mechanical contributions to a patient's strabismus. Measurements of active force indicate the magnitudes and patterns of innervation over the entire range of gaze. By comparison of these active force and passive stiffness records, nerve signal imbalances may be quantitatively distinguished from mechanical imbalances in strabismus. It is the detailed interaction of these nonlinear muscle forces and mechanical elements which determines the position of each eye in strabismus and therefore the proper surgical treatment. A brief description of actual use and a few examples of clinical results are included from over 200 human records.