Upregulated MYC expression and p53 mutations may contribute to the oncogenesis of canine Meibomian gland carcinomas.

Sebaceous carcinomas of the human ocular adnexa commonly exhibit pagetoid spread, mutations in tumor-suppressor genes, and protooncogene copy number gain. Sebaceous carcinomas are rarely reported in other species, and while the Meibomian gland (MG) represents the most common ocular adnexal structure of the canine eyelid to develop neoplasia, most are clinically and histologically benign. The objective of this study was to compare molecular features of canine MG carcinomas and adenomas. Two retrospectively identified MG carcinomas were subject to immunohistochemistry and qPCR. When compared with normal glands, MYC was upregulated in benign and malignant MG neoplasms. Aberrant p53 expression was restricted to the nuclei of intraepithelial neoplastic cells in MG carcinomas. Adipophilin expression was diminished in MG neoplasms compared with the normal MG. Our findings, if confirmed in a larger cohort of cases, could suggest that MG oncogenesis in a dog may exhibit similar molecular features as their human counterparts.

In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading.

The prognostic value of phosphatase and tensin homolog (PTEN) loss in prostate cancer has primarily been evaluated by either fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). Previously, we found that PTEN loss by IHC was associated with increased risk of upgrading from biopsy (Gleason 3 + 3) to prostatectomy (Gleason 7+). Now, using an evaluable subset of 111 patients with adjacent biopsy sections, we analyzed the association between PTEN deletion in cancer and the odds of upgrading by a highly sensitive and specific four-color FISH assay. We also compared the concordance of PTEN loss by IHC and PTEN deletion by FISH. PTEN deletion was found in 27 % (12/45) of upgraded cases compared with 11 % (7/66) of controls (P = 0.03). Cancers with PTEN deletions were more likely to be upgraded than those without deletions (adjusting for age odds ratio = 3.40, 95 % confidence interval 1.14-10.11). With respect to concordance, of 93 biopsies with PTEN protein detected by IHC, 89 (96 %) had no PTEN deletion by FISH, and of 18 biopsies without PTEN protein by IHC, 15 had homozygous or hemizygous PTEN deletion by FISH. Only 4 biopsies of the 93 (4 %) with PTEN protein intact had PTEN deletion by FISH. When the regions of uncertainty in these biopsies were systematically studied by FISH, intra-tumoral variation of PTEN deletion was found, which could account for variation in immunoreactivity. Thus, FISH provides a different approach to determining PTEN loss when IHC is uncertain. Both FISH and IHC are concordant, showing consistent positive associations between PTEN loss and upgrading.

Assessing the order of critical alterations in prostate cancer development and progression by IHC: further evidence that PTEN loss occurs subsequent to ERG gene fusion.

BACKGROUND: ERG rearrangements and PTEN (phosphatase and tensin homolog deleted on chromosome 10) loss are two of the most common genetic alterations in prostate cancer. However, there is still significant controversy regarding the order of events of these two changes during the carcinogenic process. We used immunohistochemistry (IHC) to determine ERG and PTEN status, and calculated the fraction of cases with homogeneous/heterogeneous ERG and PTEN staining in a given tumor. METHODS: Using a single standard tissue section from the index tumor from radical prostatectomies (N=77), enriched for relatively high grade and stage tumors, we examined ERG and PTEN status by IHC. We determined whether ERG or PTEN staining was homogeneous (all tumor cells staining positive) or heterogeneous (focal tumor cell staining) in a given tumor focus. RESULTS: Fifty-seven percent (N=44/77) of tumor foci showed ERG positivity, with 93% of these (N=41/44) cases showing homogeneous ERG staining in which all tumor cells stained positively. Fifty-three percent (N=41/77) of tumor foci showed PTEN loss, and of these 66% (N=27/41) showed heterogeneous PTEN loss. In ERG homogeneously positive cases, any PTEN loss occurred in 56% (N=23/41) of cases, and of these 65% (N=15/23) showed heterogeneous loss. In ERG-negative tumors, 51.5% (N=17/33) showed PTEN loss, and of these 64.7% (N=11/17) showed heterogeneous PTEN loss. In a subset of cases, genomic deletions of PTEN were verified by fluorescence in situ hybridization in regions with PTEN protein loss as compared with regions with intact PTEN protein, which did not show PTEN genomic loss. CONCLUSIONS: These results support the concept that PTEN loss tends to occur as a subclonal event within a given established prostatic carcinoma clone after ERG gene fusion. The combination of ERG and PTEN IHC staining can be used as a simple test to ascertain PTEN and ERG gene rearrangement status within a given prostate cancer in either a research or clinical setting.

Expression of the DNA-PK binding protein E4-34K fails to confer radiation sensitivity to mammalian cells.

PURPOSE: The adenovirus E4orf6 34 kDa protein (E4-34k) is known to disrupt V(D)J recombination as a result of its interaction with the catalytic subunit of cellular DNA-dependent protein kinase (DNA-PK(cs)), a major participant in the repair of DNA double-strand breaks (DSB). Previous studies have shown that cells with disrupted DSB repair and V(D)J recombination due to attenuation of DNA-PK(cs) activity exhibit a radiation-sensitive phenotype. It is not known at present whether the E4-34k protein can also modify cellular response to ionizing radiation. In an attempt to develop a novel gene therapy strategy to modify cellular radiation response, we sought to determine if expression of the adenovirus E4-34k protein resulted in sensitization to clinically relevant doses of ionizing radiation. MATERIALS AND METHODS: In order to minimize potential bias resulting from selection procedures, we performed clonogenic survival assays on DU 145 prostate cancer cells, RKO colorectal cancer cells and 293 kidney cells following transient transfection of E4-34k- and/or E1B-55k-expressing plasmids. Western blots and immunohistochemical analyses were used to demonstrate E4-34k expression within transfected cells. FACS sorting was carried out to enrich cells transfected with a plasmid that expresses both E4-34k and enhanced green fluorescent protein. RESULTS: It is shown that E4-34k expression does not affect cellular radiosensitivity of transiently transfected populations of either DU 145 prostate or RKO colon cancer cell lines. Similarly, the radiosensitivity of human embryonic kidney 293 cells, which constitutively express the E1B-55k protein, was also unaffected. The radiosensitivity of DU 145 cells co-transfected with E4-34k- and E1-55K-expressing plasmids was unchanged, suggesting that the adenovirus E1B-55k protein does not augment any effects E4-34k might have on DNA-PK(cs) activity. CONCLUSIONS: The lack of radiosensitization by E4-34k expression is quite intriguing as it is known that E4-34k interaction with DNA-PK(cs) causes disruption of V(D)J recombination, a process dependent on DSB rejoining. These data suggest that for future studies, preferential targeting of DNA-PK(cs) DSB activity will be required to influence cellular radiosensitivity.

Infected joint replacements in HIV-positive patients with haemophilia.

Joint replacement in HIV-positive patients remains uncommon, with most experience gained in patients with haemophilia. We analysed retrospectively the outcome of 102 replacement arthroplasties in 73 HIV-positive patients from eight specialist haemophilia centres. Of these, 91 were primary procedures. The mean age of the patients at surgery was 39 years, and the median follow-up was for five years. The overall rate of deep sepsis was 18.7% for primary procedures and 36.3% for revisions. This is a much higher rate of infection than that seen in normal populations. A total of 44% of infections resolved fully after medical and/or surgical treatment. The benefits of arthroplasty in haemophilic patients are well established but the rates of complications are high. As this large study has demonstrated, high rates of infection occur, but survivorship analysis strongly suggests that most patients already diagnosed with HIV infection at the time of surgery should derive many years of symptomatic relief after a successful joint replacement. Careful counselling and education of both patients and healthcare workers before operation are therefore essential.

Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor.

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.

Role of the ninaC proteins in photoreceptor cell structure: ultrastructure of ninaC deletion mutants and binding to actin filaments.

The ninaC proteins are found in Drosophila photoreceptor cells. Their primary sequences suggest they are kinase/myosin chimeras, but their myosin head-like domain is the most divergent amongst all the myosin-like proteins described to date. To investigate possible roles of the ninaC proteins in cell structure, we examined the ultrastructure of the photoreceptor cells in various ninaC mutants, and tested the ability of the proteins to interact with actin filaments in a myosin-like manner. In flies lacking the larger ninaC protein, p174, an ultrastructural phenotype was evident before eclosion. The axial actin cytoskeleton of the rhabdomeral microvilli appeared either fragmented or as an isolated structure, without linkage to the microvillar membrane. Deletion of the myosin head-like domain or the calmodulin-binding domain of p174 resulted in a similar abnormal cytoskeleton. Breakdown of the rhabdomeres followed, although at different rates depending on the deletion. Lack of the smaller protein, p132, per se did not result in photoreceptor degeneration, but in older flies there was an abnormal accumulation of multivesicular bodies. Moreover, the presence of p132 retarded the degeneration that occurs in the absence of p174, even though the p132 remained outside the rhabdomere. Biochemical studies showed that both ninaC proteins bind actin filaments and cosediment with actin filaments in an ATP-sensitive manner. These results outline structural roles for the ninaC proteins, and are consistent with the notion, suggested by their amino acid sequences, that the proteins are actin-based mechanoenzymes.

ATP causes release of intracellular Ca2+ via the phospholipase C beta/IP3 pathway in astrocytes from the dorsal spinal cord.

Calcium signaling within astrocytes in the CNS may play a role comparable to that of electrical signaling within neurons. ATP is a molecule known to produce Ca2+ responses in astrocytes, and has been implicated as a mediator of intercellular Ca2+ signaling in other types of nonexcitable cells. We characterized the signal transduction pathway for ATP-evoked Ca2+ responses in cultured astrocytes from the dorsal spinal cord. Nearly 100% of these astrocytes respond to extracellularly applied ATP, which causes release of Ca2+ from an intracellular pool that is sensitive to thapsigargin and insensitive to caffeine. We found that intracellular administration of IP3 also caused release of Ca2+ from a thapsigargin-sensitive intracellular pool, and that IP3 abolished the response to ATP. The ATP-evoked Ca2+ response was blocked by the IP3 receptor antagonist heparin, applied intracellularly, but not by N-desulfated heparin, which is not an antagonist at these receptors. The Ca2+ response caused by ATP was also blocked by a phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. Increases in [Ca2+]i were elicited by intracellular application of activators of heterotrimeric G-proteins, GTP gamma S and AIF4-. On the other hand, [Ca2+], was unaffected by a G-protein inhibitor, GDP beta S, but it did abolish the Ca2+ response to ATP. Pretreating the cultures with pertussis toxin did not affect responses to ATP. Our results indicate that in astrocytes ATP-evoked release of intracellular Ca2+ is mediated by IP3 produced as a result of activating phospholipase C coupled to ATP receptors via a G-protein that is insensitive to pertussis toxin. ATP is known to be released under physiological and pathological circumstances, and therefore signaling via the PLC-IP3 pathway in astrocytes is a potentially important mechanism by which ATP may play a role in CNS function.

ATP-evoked increases in intracellular calcium in neurons and glia from the dorsal spinal cord.

ATP has been proposed as a possible chemical mediator of synaptic transmission in the spinal dorsal horn on the basis that it is released in dorsal horn synaptosomes in a Ca(2+)-dependent manner and that its effects mimic those of synaptic inputs to dorsal horn neurons. In the present study we examined the actions of ATP on neurons and glia in cell culture using optical and electrophysiological recording techniques. We found that ATP increased intracellular Ca2+ concentration ([Ca2+]i) in > 99% of astrocytes. In contrast, only 35% of neurons and 20% of oligodendrocytes responded to ATP. The prevalence of the ATP-evoked response in astrocytes led us to characterize the type of receptor mediating the response, the source of Ca2+, and the membrane currents activated by ATP. We found that ADP was approximately equipotent with ATP in increasing [Ca2+]i whereas AMP and adenosine had no effect. In addition, responses to ATP were blocked in a concentration-dependent manner by the P2 purinergic receptor antagonist suramin. Furthermore, as it was found that 2-methylthio-ATP was more potent than ATP and that beta, gamma-methylene-ATP was ineffective, the responses were mediated via the P2 gamma subtype of purinergic receptor. The increase in [Ca2+]i evoked by ATP persisted in extracellular medium with no added Ca2+ and containing EGTA, indicating that this increase was due to release of Ca2+ from intracellular stores. Release of Ca2+ by ATP was blocked by thapsigargin but was unaffected by caffeine. ATP had several effects on membrane current activating inward, outward, and mixed currents despite uniformly causing increases in [Ca2+]i. These observations indicate that ATP has diverse electrophysiological effects on astrocytes as well as increasing [Ca2+]i in these cells. We speculate that ATP released from synaptic terminals in the dorsal horn might act not only on postsynaptic neurons but also on perisynaptic astrocytes. Thus, a physiological role for ATP may be as a neuronal-glial signaling molecule within the spinal dorsal horn.

Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells.

The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.

Distribution of the myosin I-like ninaC proteins in the Drosophila retina and ultrastructural analysis of mutant phenotypes.

The Drosophila ninaC gene encodes for two head-specific proteins of 132 kDa and 174 kDa. Their predicted amino acid sequences indicate that they may have myosin I and kinase properties. We have: (1) determined the cellular and subcellular distributions of the ninaC proteins in the Drosophila retina by electron microscopic immunocytochemistry with an antibody specific for epitopes shared by both proteins; (2) characterized the ultrastructure of the mutant phenotype. The proteins were detected only in the photoreceptor cells, but were detected in all classes of the compound eye photoreceptors. Within the photoreceptors, they were found in the rhabdomeral microvilli and the cytoplasm adjacent to the rhabdomeres. This distribution coincides with that shown previously for actin filaments. Immunolabelling of tissue from the ninaC P221 mutant, which lacks the 174 kDa protein, and two mutants whose rhabdomeres degenerate, suggests that the 132 kDa protein is present primarily in the cytoplasm adjacent to the rhabdomeres, and that the 174 kDa protein is concentrated in the rhabdomeres. Our ultrastructural analysis showed that the axial cytoskeleton of the rhabdomeral microvilli (which contains filamentous actin) was absent in both the null and P221 mutants. In the photoreceptor cell cytoplasm, the number of multivesicular bodies in the null mutant, but not the P221 mutant, was 3-fold greater in comparison with wild-type.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of oral Candida tropicalis infection to systemic candidiasis in a patient with leukemia.

Oropharyngeal candidiasis is an extremely common complication in patients receiving chemotherapy for leukemia. Candida tropicalis appears to be the major infectious agent when these patients develop candidemia. In this article, a case of C tropicalis fungemia with oropharyngeal manifestations is presented. The relationship of oropharyngeal candidiasis to oral candidal infection is discussed.

Bacterial endocarditis of dental origin: report of case.

Although appreciated by most practitioners, the fact that dental infection may be the source of bacteremia without a history of recent dental procedures is occasionally overlooked. The case reported here illustrates what we feel is an example of such a phenomenon. The eradication of the oral foci of infection enhanced the patient's response to therapy and prompted his ultimate recovery.