A teratologic suppressor role for p53 in benzo[a]pyrene-treated transgenic p53-deficient mice.

DNA damage may mediate birth defects caused by many drugs and environmental chemicals, therefore p53, a tumour suppressor gene that facilitates DNA repair, may be critically embryoprotective. We have studied the effects of the environmental teratogen, benzo[a]pyrene, on pregnant heterozygous p53-deficient mice. Such mice exhibited between 2- to 4-fold higher embryotoxicity and teratogenicity than normal p53-controls. Fetal resorptions reflecting in utero death were genotyped using the polymerase chain reaction and found to be increased 2.6-fold and 3.6-fold respectively with heterozygous and homozygous p53-deficient embryos. These results provide the first direct evidence that p53 may be an important teratological suppressor gene which protects the embryo from DNA-damaging chemicals and developmental oxidative stress.

Mucinous ovarian cancer.

Mucinous epithelial ovarian cancer (mEOC) accounts for approximately 10% of EOCs. Patients presenting with early-stage disease have an excellent prognosis, however, those with advanced disease have a poor outcome with relative resistance to standard ovarian cancer chemotherapy. Molecular and genetic studies demonstrate differences between mucinous and serous EOC supporting the concept that these tumors develop along separate pathways. Together with the observed differences in clinical behavior and outcome for mEOC, there is a need to develop specific therapeutic strategies for this histologic subtype. The relative rarity of advanced mEOC has resulted in few patients enrolled in major ovarian cancer trials. The results of such trials may not necessarily reflect those specific to mEOC. Separate trials testing alternative chemotherapeutics are required. Metastatic mucinous tumors from other sites such as the gastrointestinal tract may present with ovarian involvement. For all mucinous tumors of the ovary, establishing primary as opposed to metastatic cancers is important. Clinical presentation, tumor markers, histologic, and immunohistochemical features are helpful in distinguishing most cases.

Management of metastatic gastrointestinal stromal tumour in the Glivec era: a practical case-based approach.

Gastrointestinal stromal tumour is now recognized as a distinct pathological malignancy and has received much attention over the last few years. Despite almost universal resistance to chemotherapy, a novel therapy, Imatinib, which targets the KIT receptor, has changed the natural history of this disease. We have audited the first 26 consecutive patients with gastrointestinal stromal tumour treated over 4 years at a single institution. A practical guide to the management of common toxicities and drug resistance is reported with a review of the published reports. Many of the strategies used are likely to be widely applicable to the use of targeted therapies in other malignancies.

Induction of a substrate for casein kinase II during lymphocyte mitogenesis.

Particulate fractions prepared from concanavalin A-activated murine T lymphocytes contain an endogenous protein kinase that phosphorylates an endogenous protein substrate of Mr 112 000. The phosphorylation of 112 kDa protein is greatly reduced or absent in unstimulated T cells. Phosphoamino acid analysis indicates that 112 kDa protein is labeled on a serine. Add-back experiments using purified protein kinases indicate that 112 kDa protein serves as a substrate for casein kinase II. Phosphorylation of 112 kDa protein by the endogenous kinase is inhibited by heparin, a known casein kinase II inhibitor. The site or sites modified by the endogenous kinase and exogenous casein kinase II appear identical by peptide-mapping experiments. A time-course of the appearance of phosphorylated 112 kDa protein following stimulation with concanavalin A, measured in the presence or absence of added casein kinase II, suggests that 112 kDa protein is induced in activated T cells. Subcellular localization studies suggest that 112 kDa protein is a nuclear protein. Silver-binding and purification studies suggest that 112 kDa protein is of the nucleolar organizing region.

Properties of a tyrosine protein kinase from calf thymus. Response to ionic strength and divalent cations.

A tyrosine protein kinase activity has been partially purified from calf thymus using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Detergent extracts of calf thymus possessed only low levels of specific peptide phosphorylating activity when assayed at low ionic strength. The inclusion of NaCl at a concentration of 2 M stimulated endogenous tyrosine protein kinase activity, while the activity of other endogenous kinases was inhibited. This sensitivity to NaCl was retained following partial purification of the enzyme. The phosphorylation of other substrates such as casein or the R-R-SRC peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) by the tyrosine protein kinase was less sensitive to NaCl. Phosphorylation of the PK-1 peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) by the purified catalytic subunit of cAMP-dependent protein kinase was inhibited by NaCl. The effect of NaCl on angiotensin I phosphorylation could be mimicked by KCl or sodium acetate. The principal effect of NaCl was to increase the Vmax of the enzyme for the phosphorylation of angiotensin I. At low ionic strength, Mn2+ and Co2+ were the preferred required divalent cations. At elevated NaCl concentrations Mg2+ was preferred, with half-maximal activation occurring at 35 mM Mg2+. By conducting peptide phosphorylation assays in the presence of elevated levels of Mg2+ and NaCl, tyrosine protein kinase activity can readily be detected in extracts from cell lines that express low levels of the enzyme.

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk.

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Preliminary X-ray analysis of crystals of murine adenosine deaminase.

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.

Oxidative stress and thiol depletion in plasma and peripheral blood lymphocytes from HIV-infected patients: toxicological and pathological implications.

OBJECTIVES: To determine, first, whether the plasma and lymphocytes of HIV-positive individuals and AIDS patients have alterations in the major thiols glutathione and cysteine, and/or their oxidative disulphide and mixed disulphide products; and, secondly, whether thiol/disulphide status differs in patients with sulphonamide drug hypersensitivity reactions. DESIGN: Thiols provide critical cellular defence against toxic drug reactive intermediates and endogenous oxidative stress, and may modulate HIV replication. Glutathione is reported to be low in HIV-positive individuals and AIDS patients, but this is controversial and the mechanism responsible is unknown. Also unknown is whether altered thiol/disulphide status determines the predisposition of HIV-positive and AIDS patients to drug reactions. METHODS: Thiols and disulphides were measured by high-performance liquid chromatography. RESULTS: Both plasma thiols were decreased by approximately 58% in HIV-positive individuals and AIDS patients compared with uninfected controls (P < 0.05), with increases of up to threefold in oxidized products (P < 0.05). Similarly, in lymphocytes, thiols were decreased by 30-35% (P < 0.05), with apparent increases in oxidized products. For both glutathione and cysteine, the thiol/disulphide ratios also were decreased (P < 0.05). The plasma and lymphocyte glutathione thiol/disulphide ratios were highly correlated (r = 0.7661; P = 0.0001) among all subjects. No parameters differed in patients with drug reactions, or with antiretroviral therapy. CONCLUSIONS: The enhanced thiol oxidation in HIV-positive individuals and AIDS patients indicates oxidative stress, which also contributes to thiol depletion, and may enhance damage to macromolecular targets. These mechanisms may contribute to enhanced viral replication and other pathological outcomes. HIV-positive individuals' and AIDS patients' predisposition to drug hypersensitivity reactions appears to be unrelated to thiol/disulphide status.

Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1.

Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.

Acute and late toxicity associated with sequential bleomycin-containing chemotherapy regimens and radiation therapy in the treatment of carcinoma of the nasopharynx.

Between 1975 and 1984, 33 patients with squamous cell carcinoma of the nasopharynx received adjuvant chemotherapy before and/or after definitive radiotherapy at UT M. D. Anderson Hospital. The favored chemotherapy regimens during this time were BCMF (bleomycin, cyclophosphamide, methotrexate, and 5-FU) and PMB (cisplatinum, methotrexate, and bleomycin). Total radiation doses to the primary site averaged 65 Gy for T1 and T2 lesions and 70 Gy for T3 and T4 lesions. Neck nodes were given boost treatments to a maximum of 70 Gy, depending on the extent of the disease. The outcome of treatment in these patients was compared to that of a stage-matched group of 71 patients treated during the same time period with radiotherapy alone. However, the groups were not matched with regard to histologic subtypes: 45% of the radiation-only group had prognostically unfavorable keratinizing squamous carcinomas (WHO 1) compared with 18% of the combined modality group. Overall disease-free survival at 5 years was 63% in the combined modality group and 44% in the radiation only group (p = 0.15). Both acute reactions and late treatment complications were much more frequent and severe in patients receiving combined modality treatment. In patients treated with chemotherapy prior to radiation therapy, 10/20 (50%) experienced severe acute toxicity (RTOG Grade 3 or 4) versus 9/71 (13%) in the radiotherapy-only group. Severe late normal tissue injury occurred in 15/33 (45%) of the combined modality group versus 5/71 (7.0%) in the control group. The majority of the late complications in the adjuvant chemotherapy group consisted of severe soft tissue and muscle fibrosis. The average total bleomycin dose in the patients with severe late soft tissue and muscle fibrosis was 336 mg. The actuarial risk of developing a severe late complication by 2 years after treatment was 68% in the combined modality group versus 8% in the radiation-therapy-only group (p = .001). The probability of remaining both disease-free and complication-free at 5 years was 40% in the radiation-only group and 22% in the combined-modality group (p = 0.08). Comparison of these results with other published reports emphasizes the importance of late toxicity data in assessing the ultimate value of combined modality therapy.

The Annonaceous acetogenin bullatacin is cytotoxic against multidrug-resistant human mammary adenocarcinoma cells.

Cytotoxic effects of the Annonaceous acetogenin, bullatacin, were studied in multidrug-resistant (MDR) human mammary adenocarcinoma (MCF-7/Adr) cells vs. the parental non-resistant wild type (MCF-7/wt) cells. Bullatacin was effectively cytotoxic to the MCF-7/Adr cells while it was more cytostatic to the MCF-7/wt cells. ATP depletion is the mode of action of the Annonaceous acetogenins, and these agents offer a special advantage in the chemotherapeutic treatment of MDR tumors that have ATP-dependent mechanisms.

Carcinoma in diverticulum of female urethra.

A case of carcinoma originating in a diverticulum of the urethra in a female patient is presented. A review of 143 cases of carcinoma of the female urethra treated from 1948 to 1984 at The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston disclosed 6 additional patients with diverticular carcinoma. Analysis of their clinical features, treatments--various combinations of primary excision, radiotherapy, and chemotherapy--and survival results indicate that survival is primarily a function of grade. Only 40 cases of carcinoma in urethral diverticula are recorded in the world literature. The majority are adenocarcinomas, and the most frequent presenting symptoms are dysuria, frequency, and urgency. Radiotherapy successfully established long-term control of the disease with low morbidity in all of our patients who had low-grade tumors.

Angiographically cryptic AVM presenting as a pontine tumor. Case report.

A 19-year-old child became disoriented and ataxic 6 days after a diphtheria-pertussis-tetanus (DPT) booster injection. Computerized tomographic scan revealed a hyperdense mesencephalic-pontine mass, and angiography demonstrated an avascular mass. Pontine biopsy revealed no abnormality. Her clinical course fluctuated until her death 15 months later. At autopsy, an angiographically cryptic arteriovenous malformation (AVM) was present in the mesencephalon and pons. Vascular malformations of the brain stem producing prolonged clinical courses, are reviewed.

A review of screening mammography participation and utilization in Canada.

INTRODUCTION: Participation rate is an important indicator for a screening program's effectiveness; however, the current approach to measuring participation rate in Canada is not comparable with other countries. The objective of this study is to review the measurement of screening mammography participation in Canada, make international comparisons, and propose alternative methods. METHODS: Canadian breast cancer screening program data for women aged 50 to 69 years screened between 2004 and 2006 were extracted from the Canadian Breast Cancer Screening Database (CBCSD). The fee-for-services (FSS) mammography data (opportunistic screening mammography) were obtained from the provincial ministries of health. Both screening mammography program participation and utilization were examined over 24 and 30 months. RESULTS: Canada's screening participation rate increases from 39.4% for a 24-month cut-off to 43.6% for a 30-month cut-off. The 24-month mammography utilization rate is 63.1% in Canada, and the 30-month utilization rate is 70.4%. CONCLUSION: Due to the differences in health service delivery among Canadian provinces, both programmatic participation and overall utilization of mammography at 24 months and 30 months should be monitored.

Aerobic production of isoamyl acetate by overexpression of the yeast alcohol acetyl-transferases AFT1 and AFT2 in Escherichia coli and using low-cost fermentation ingredients.

Isoamyl acetate, produced via fermentation, is a natural flavor chemical with applications in the food industry. Two alcohol acetyltransferases from Saccharomyces cerevisiae (ATF1 and ATF2) can catalyze the esterification of isoamyl alcohol with acetyl coenzyme A. The respective genes were cloned and expressed in an appropriate ack-pta(-) strain of Escherichia coli. The engineered strains produce isoamyl acetate when isoamyl alcohol is added to the culture medium. Aerobic shake flask experiments examined isoamyl acetate production over various growth times, temperatures, and initial optical densities. The strain carrying the pBAD-ATF1 plasmid exhibited a high molar ester yield from glucose (1.13) after 48 h of aerobic growth at 25 degrees C. Low-cost media components, such as fusel oil, sorghum glucose and corn steep liquor, were found to give a high yield of isoamyl acetate. High-cell-density gave an increased isoamyl acetate yield of 0.18 g/g of glucose consumed.

Small cell of the ovary, hypercalcemic type -- analysis of combined experience and recommendation for management. A GCIG study.

UNLABELLED: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare tumor typically affecting young women. It is an aggressive malignancy with a poor prognosis and few long-term survivors. OBJECTIVE: Investigate the outcome of patients with SCCOHT. METHOD: Data were collected for patients with SCCOHT treated in Australia, Canada and Europe. Information included stage, surgery, chemotherapy, radiotherapy, recurrence and survival. RESULTS: The median follow-up is 13 months for all patients and 35.5 months in surviving patients. Ten patients had FIGO stage I tumors, six stage III tumors and one stage unknown. All underwent surgical resection. Adjuvant platinum-based chemotherapy was given to all patients. Seven received adjuvant radiotherapy with either pelvic and para-aortic radiotherapy, average dose 46.5 Gy (40 Gy/25# - 50.4 Gy/23#), or pelvic and whole abdominal radiotherapy, average dose 45 Gy to pelvis and 25 Gy (22.5 Gy/22# - 30 Gy/25#) to abdomen. The median survival for stage I tumors was not reached and was 6 months for stage III tumors. For the ten patients with stage I tumors: six received adjuvant radiotherapy with five alive and disease-free; four received no adjuvant radiotherapy with one alive and disease-free, while three have relapsed with one alive and disease-free after resection. Of the seven patients with stage III or unknown stage tumors, all but one have died. Recurrences were most frequent in the pelvis and the abdomen. Patients receiving salvage treatment with chemotherapy and radiotherapy did poorly. CONCLUSION: We advocate a multi-modality treatment approach including surgery, chemotherapy with the addition of radiotherapy either sequentially or concurrently.

Social implications of gummivory in marmosets.

Gummivory or exudate feeding is a major dietary specialization which has received relatively little attention in the literature. While plant exudates contribute to the diet of many primate species, we suggest that the callitrichid species Cebuella pygmaea and Callithrix jacchus are obligate exudate feeders under free-ranging conditions. Callithrix jacchus provides an excellent model for examining the effects of exudate feeding and foraging upon social behavior, since other callitrichid species of similar body size do not share this dietary specialization. We review the effects of exudate foraging on specific social behaviors observed both in field and laboratory populations of C. jacchus. By comparing this species to closely related species, exudate foraging is seen to (1) be retained under laboratory conditions, (2) increase the frequency of territorial marking behavior while decreasing the frequency of overt aggression in males, (3) decrease the duration of infant care, and (4) increase the number of nonadults in social groups but not affect group size. The evidence presented supports the hypothesis that the consequences of exudate foraging in C. jacchus are fundamental and socially complex.

Detection of a novel lymphocyte protein-tyrosine kinase by renaturation in polyacrylamide gels.

Protein kinase activity, including activity specific for the phosphorylation of tyrosine residues, can be detected among particulate fraction proteins of T cell lymphomas after separation by SDS-polyacrylamide gel electrophoresis. Putative protein kinases are detected by renaturation of enzyme activity directly within the gel following removal of detergent. LSTRA, a cell line that exhibits elevated levels of protein-tyrosine kinase activity, was found to express a predominant protein-tyrosine kinase of molecular weight 30,000. This same enzyme was present in T lymphocytes and other T lymphoid cell lines. Studies involving rapid preparation of protein fractions, limited proteolysis and one-dimensional peptide mapping did not demonstrate a direct relationship between the phosphorylated 30,000 dalton protein and the predominant 56,000 dalton phosphotyrosine containing protein that is observed following phosphorylation of LSTRA cell particulate fractions in vitro.

Kin preference in marmosets and tamarins: Saguinus oedipus and Callithrix jacchus (Callitrichidae, primates).

Field studies on callitrichid primates have suggested that some species may exhibit a communal breeding system in which two or more males mate with a single female and cooperatively participate in infant rearing. However, high levels of aggression have been observed between males under both laboratory and field conditions. In avian models for communal breeding, cooperating males are often closely related. In a laboratory test of a hypothesis of the amelioration of aggression based on kinship, four mated pairs of both Saguinus oedipus and Callithrix jacchus were introduced to both novel male conspecifics and to their own male siblings, from whom they had been separated for a minimum of 4 months. Significant differences (P less than .01) were seen in the responses of the two species to male conspecifics generally: S. oedipus males responded to intruders with overt aggression significantly more frequently than did marmosets while C. jacchus males more frequently scent-marked than did tamarins. Evidence of preference for kin males was seen in the significantly decreased frequencies of these behaviors in the presence of kin males over those observed in the presence of novel males. It is argued that, under field conditions, cooperation between male callitrichids may be facilitated by kinship. Further, twin births, short gestation lengths, and interbirth intervals seen in callitrichid primates all contribute to the number of socially familiar kin with which an individual animal might cooperate.