Protein kinase Cδ is required for ErbB2-driven mammary gland tumorigenesis and negatively correlates with prognosis in human breast cancer.

Protein kinase C δ (PKCδ) regulates apoptosis in the mammary gland, however, the functional contribution of PKCδ to the development or progression of breast cancer has yet to be determined. Meta-analysis of ErbB2-positive breast cancers shows increased PKCδ expression, and a negative correlation between PKCδ expression and prognosis. Here, we present in-vivo evidence that PKCδ is essential for the development of mammary gland tumors in a ErbB2-overexpressing transgenic mouse model, and in-vitro evidence that PKCδ is required for proliferative signaling downstream of the ErbB2 receptor. Mouse mammary tumor virus (MMTV)-ErbB2 mice lacking PKCδ (δKO) have increased tumor latency compared with MMTV-ErbB2 wild-type (δWT) mice, and the tumors show a dramatic decrease in Ki-67 staining. To explore the relationship between PKCδ and ErbB2-driven proliferation more directly, we used MCF-10A cells engineered to express a synthetic ligand-inducible form of the ErbB2 receptor. Depletion of PKCδ with short hairpin RNA inhibited ligand-induced growth in both two-dimensional (2D) (plastic) and three-dimensional (3D) (Matrigel) culture, and correlated with decreased phosphorylation of the ErbB2 receptor and reduced activation of Src and MAPK/ERK pathways. Similarly, in human breast cancer cell lines in which ErbB2 is overexpressed, depletion of PKCδ suppresses proliferation, Src and ERK activation. PKCδ appears to drive proliferation through the formation of an active ErbB2/PKCδ/Src signaling complex, as depletion of PKCδ disrupts association of Src with the ErbB2 receptor. Taken together, our studies present the first evidence that PKCδ is a critical regulator of ErbB2-mediated tumorigenesis, and suggest further investigation of PKCδ as a target in ErbB2-positive breast cancer.

The challenge of early surgery for cryptorchidism.

OBJECTIVE: In congenital cryptorchidism, a recent Nordic Consensus report recommends surgical correction at 6-12 months of age to prevent male infertility. In published series of orchiopexies the median age at surgery is often 3 years or more. The aim of this study was to evaluate whether early surgery is technically safe. MATERIAL AND METHODS: The study included 356 boys with 418 orchiopexies and median 1 year of follow-up. RESULTS: At follow-up 367 testes were in the scrotum without iatrogenic atrophy. Eight testes were atrophied and in 43 cases a redo operation was performed to achieve a scrotal position of the testis. All acquired undescended testes had a successful result. The age at operation for the group with congenital undescended testes in the intracanalicular position or in a position close to the external inguinal annulus was 4 months to 14.5 years. In the latter group the median age at operation for the 41 failures was 2 years and 4 months, which is significantly younger than the median age for the more successful operations (3 years and 9 months). CONCLUSION: The standard orchiopexy is technically demanding in small boys. Focus on successful operative results in specialist centres is important when treating cryptorchidism with early surgery, otherwise the positive beneficial biological impact on fertility potential may be lost due to treatment failure.

Loss of protein kinase C delta alters mammary gland development and apoptosis.

As apoptotic pathways are commonly deregulated in breast cancer, exploring how mammary gland cell death is regulated is critical for understanding human disease. We show that primary mammary epithelial cells from protein kinase C delta (PKCδ) -/- mice have a suppressed response to apoptotic agents in vitro. In the mammary gland in vivo, apoptosis is critical for ductal morphogenesis during puberty and involution following lactation. We have explored mammary gland development in the PKCδ -/- mouse during these two critical windows. Branching morphogenesis was altered in 4- to 6-week-old PKCδ -/- mice as indicated by reduced ductal branching; however, apoptosis and proliferation in the terminal end buds was unaltered. Conversely, activation of caspase-3 during involution was delayed in PKCδ -/- mice, but involution proceeded normally. The thymus also undergoes apoptosis in response to physiological signals. A dramatic suppression of caspase-3 activation was observed in the thymus of PKCδ -/- mice treated with irradiation, but not mice treated with dexamethasone, suggesting that there are both target- and tissue-dependent differences in the execution of apoptotic pathways in vivo. These findings highlight a role for PKCδ in both apoptotic and nonapoptotic processes in the mammary gland and underscore the redundancy of apoptotic pathways in vivo.

Unchanged incidence and increased survival in children with neuroblastoma in Denmark 1981-2000: a population-based study.

Treatment results for neuroblastoma in Denmark have been poorer than in other Nordic countries, so we investigated whether a change in incidence, stage distribution and survival had occurred between 1981 and 2000. Clinical data were retrieved from the medical charts of 160 children <15 years of age with extra-cranial neuroblastoma (n=139) or ganglioneuroblastoma (n=21) diagnosed in Denmark between 1981 and 2000. The minimal follow-up time was 52 months. Statistical analyses were performed in STATA. The incidence was 8.55 per million children below 15 years of age (world standard 9.6) and 42.6 per million children below 12 months of age, and it has remained unchanged since 1970. The median age at diagnosis was 27 months. In all, 32% of the children were aged below 12 months at diagnosis, 53% had metastatic disease and in 12% the diagnosis was made incidentally. Prognostic factors such as age, stage and site of primary tumour were the same as in other studies and did not change. During the study period, the mortality rate decreased steadily, and the 5-year survival rate increased from 38% in 1981-1985 to 59% in 1996-2000, corresponding to the level found in other Western countries. Increased survival was also seen in children with metastatic disease. Participation in international studies, better supportive care and possibly postoperative autologous stem cell transplantation may have contributed to the increased survival.

Juvenile nasopharyngeal angiofibromas in Denmark 1981-2003: diagnosis, incidence, and treatment.

CONCLUSIONS: Juvenile nasopharyngeal angiofibroma (JNA) is a rare tumor in young males, with a non-negligible potential for recurrence. Preoperative embolization is a safe procedure that diminishes the peroperative blood loss and the need for blood transfusion. The endoscopic approach was used with good results in JNA stage I and II (Chandler). OBJECTIVES: To estimate the incidence rate of JNA in the Danish population and to describe symptoms and treatment. PATIENTS AND METHODS: This was a national retrospective cohort study. All cases of JNA diagnosed in Denmark from 1981 to 2003 were identified. Data were extracted from medical records. RESULTS: Forty-five male (no female) JNA cases were identified. In 43 cases, clinical data were recovered. Median age was 15 years. The incidence rate in Denmark was 0.4 cases per million inhabitants per year and 3.7 cases per million males (aged 10-24) per year. All patients underwent surgery, and the endoscopic approach was increasingly being used. The embolization procedure proved to be safe and decreased the intraoperative blood loss statistically to 650 ml in the embolized group from an average of 1200 ml in the non-embolized group (p<0.05). Similarly, the need for peroperative blood transfusion was reduced (p<0.005). The primary recurrence rate was 23% and no patients died.

The relation of germ cells per tubule in testes, serum inhibin B and FSH in cryptorchid boys.

At bilateral orchiopexy bilateral biopsies may be indicated to determine fertility potential. It is currently unknown if the serum inhibin B levels at time of orchiopexy reflect the testicular status of the bilaterally cryptorchid child. The aim of this study was to relate the results of inhibin B and FSH measurements with testicular biopsy parameters in bilateral cryptorchid boys. Included were 25 boys with bilateral cryptorchidism, median 2.5 years (9 months to 5.5 years) at surgery for bilateral cryptorchidism with simultaneous testicular biopsies, and blood sample for inhibin B and FSH. The number of spermatogonia and gonocytes was measured in 100 tubular transverse sections, the S/T and the mean-S/T of the patient was found, and expressed as percent of lowest normal value. Inhibin B and FSH were measured and related to age-specific values. Forty percent (10/25) of the patients had very low mean-S/T (mean-S/T<10% of lowest normal-value). Inhibin B was decreased in 24% (6/25) of the patients, all with decreased mean-S/T, predominantly with a mean-S/T<10% of lowest normal-value (p<0.05). There was a negative correlation between inhibin B and FSH (p<0.05). In cases of mean-S/T<10% of lowest normal-value and decreased inhibin B, we found increased FSH in 9% (2/22) of the patients and hypergonadotropic hypogonadism was suspected. Low FSH was found in 5% (1/22) of the patients and hypogonadotropic hypogonadism was suspected. Low inhibin B predicts a serious condition in respect of infertility. Low FSH and inhibin B indicates examination for hypogonadotropic hypogonadism. In bilateral cryptorchid boys inhibin B levels correlated negatively to FSH.

The incidence of bilateral cryptorchidism is increased and the fertility potential is reduced in sons born to mothers who have smoked during pregnancy.

PURPOSE: Recent studies have demonstrated a high prevalence of cryptorchidism, decreasing semen quality and increasing incidence of testicular cancer. These changes seem to be interrelated, and may be symptoms of a common underlying entity with foundations in fetal life. We investigated the influence of maternal smoking on fertility status in offspring cryptorchidism. MATERIALS AND METHODS: We prospectively studied consecutive patients presenting to the pediatric surgery department between 1996 and 2005. A total of 157 boys 1 to 5.9 years old underwent surgery for cryptorchidism with simultaneous testicular biopsy, and exhibited well preserved testicular parenchyma. Only white patients with Danish-speaking mothers who had reported pregnancy history including smoking habits during pregnancy and history of the offspring were included. The patients had cryptorchidism only and none received hormonal treatment before surgery. The number of spermatogonia and gonocytes per tubule cross-section was assessed and compared to normal values from autopsy material. RESULTS: The group of boys with cryptorchidism whose mothers had smoked heavily during pregnancy (ie more than 10 cigarettes daily throughout the pregnancy) had a significantly increased risk of bilateral cryptorchidism (52%, or 11 of 21 patients), and a decreased number of spermatogonia and gonocytes per tubule cross-section, which was absolute (0.097 [0 to 0.75]) and age related (14% [0% to 198%] of normal for age) compared to boys whose mothers did not smoke (20%, or 22 of 112 patients, 0.140 [0 to 2.14] and 37% [0% to 563%] of normal for age, p <0.01, p <0.05 and p <0.05, respectively). CONCLUSIONS: A close relationship between maternal smoking during pregnancy and adverse trends in offspring reproductive health in relation to cryptorchidism was observed.

Erroneous genetic sex determination of a newborn twin girl due to chimerism caused by foetal blood transfusion. A case report.

OBJECTIVE: We present a case of erroneous sex determination in a newborn twin girl (twin A) due to chimerism. CASE REPORT: Amniocentesis and ultrasound examination had pointed towards male sex of both twins. At birth, twin A presented as a phenotypically normal female with 46,XY karyotype, and 46,XY gonadal dysgenesis was suspected. Twin B was a normal male. RESULTS: In our department, further examinations of twin A included undetectable testosterone and inhibin-B and elevated FSH. Ultrasound suspected an infantile uterus, and sequencing of the SRY gene was normal. After gonadectomy, a 46,XX karyotype was demonstrated in both normal infantile ovaries and in the fibroblasts from a skin biopsy. Analysis of X-linked markers in DNA from blood lymphocytes in both twins was identical, consistent with 46,XY karyotypes. CONCLUSION: Twin A is a 46,XX female with a chimeric 46,XY blood cell line due to intrauterine transfusion from her twin brother.

High expression of markers of apoptosis in Langerhans cell histiocytosis.

AIMS: Langerhans cell histiocytosis is a rare disease with clonal proliferation of dendritic histiocytes, occurring most frequently in infancy and early childhood. In the localized form (single system), the disease is self-limiting, but in the cases of multisystem disease a third of the patients develop organ dysfunction. In these cases the prognosis is poor. Our objective has been to study the immunohistochemical expression of Fas and Fas-ligand (Fas-L) in order to determine whether the level of expression of these proteins could predict the outcome of the disease. We also wanted to determine the number of apoptotic cells to compare with the expression of Fas and Fas-L. METHODS AND RESULTS: We analysed the expression of Fas and Fas-L in 76 infiltrates from 49 paediatric patients with Langerhans cell histiocytosis. We also compared the results with the expression of the tumour suppressor protein p53 and the number of cells in apoptosis detected with TUNEL. Langerhans cell histiocytosis cells showed strong expression of p53 and in some cases co-expression of Fas and Fas-L. The expression of Fas-L was significantly higher in infiltrates from patients with single-system disease. The actual number of pathological Langerhans cells in apoptosis as estimated by TUNEL was low. CONCLUSIONS: The low number of TUNEL-reactive cells can be explained by the rapid turnover of apoptotic cells in the tissue, not leaving the apoptotic cells long enough in the tissue to be detected. The co-expression of Fas and Fas-L in some Langerhans cells can lead to an autocrine apoptotic shortcut, mediating the death of the double-positive cells. Our findings suggest that apoptosis mediated through the Fas/Fas-L pathway may contribute to the spontaneous regression of lesions in single-system disease. A delicate balance between autocrine death and survival of Langerhans cells may have been disturbed in patients with multisystem lesions.

Biosynthesis and metabolic engineering of glucosinolates.

Glucosinolates are amino acid-derived natural plant products found throughout the Capparales order. Glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi. The conversion of amino acids to aldoximes is a key step in glucosinolate biosynthesis. This step is catalyzed by cytochromes P450 from the CYP79 family. The post-aldoxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional group and low substrate-specificity for the side chain. Therefore, we have been able to metabolically engineer new glucosinolate profiles into Arabidopsis by altering the levels of endogenous CYP79s and by introducing exogenous CYP79s. The approach has great potential for design of metabolically engineered plants with improved pest resistance and increased nutritional value.

Long-distance phloem transport of glucosinolates in Arabidopsis.

Glucosinolates are a large group of plant secondary metabolites found mainly in the order Capparales, which includes a large number of economically important Brassica crops and the model plant Arabidopsis. In the present study, several lines of evidence are provided for phloem transport of glucosinolates in Arabidopsis. When radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) and sucrose were co-applied to the tip of detached leaves, both tracers were collected in the phloem exudates at the petioles. Long-distance transport of [(14)C]p-OHBG was investigated in wild-type and transgenic 35S::CYP79A1 plants, synthesizing high amounts of p-OHBG, which is not a natural constituent of wild-type Arabidopsis. In both wild-type and 35S::CYP79A1 plants, radiolabeled p-OHBG was rapidly transported from the application site into the whole plant and intact p-OHBG was recovered from different tissues. The pattern of distribution of the radioactivity corresponded to that expected for transport of photoassimilates such as sucrose, and was consistent with translocation in phloem following the source-sink relationship. Radiolabeled p-OHBG was shown to accumulate in the seeds of wild-type and 35S::CYP79A1 plants, where p-OHBG had been either exogenously applied or endogenously synthesized from Tyr in the leaves. p-OHBG was found in phloem exudates collected from cut petioles of leaves from both wild-type and 35S::CYP79A1 plants. Phloem exudates were shown to contain intact glucosinolates, and not desulphoglucosinolates, as the transport form. It is concluded that intact glucosinolates are readily loaded into and transported by the phloem.

Characterization of transgenic Arabidopsis thaliana with metabolically engineered high levels of p-hydroxybenzylglucosinolate.

The cytochrome P450 CYP79A1 catalyzes the conversion of L-tyrosine to p-hydroxyphenylacetaldoxime, the first step in the biosynthetic pathway of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. We have demonstrated that introduction of CYP79A1 into Arabidopsis thaliana (L.) Heynh. results in the production of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate (p-OHBG), not found in wild-type A. thaliana (Bak et al., 1999, Plant J. 20: 663 671). In the present study, glucosinolate profiles and contents in various tissues (roots, leaves, stems, closed flower buds and green siliques) of A. thaliana plants expressing CYP79A1 were analyzed by high-performance liquid chromatography. The total glucosinolate content in these tissues was increased 3.5- to 4.5-fold in comparison with the level of the control plants. The increase was due solely to the production of p-OHBG, as the composition of the major endogenous aliphatic and indole glucosinolates was not affected. Conversely, in mature seeds the total glucosinolate content of CYP79A1 and control plants was similar, with p-OHBG accounting for ca. 30%. The transcript level of the postoxime enzyme UDP-glucose:thiohydroximate glucosyltransferase in leaves of CYP79A1 plants was increased ca. 50% compared with control plants, indicating that the post-oxime enzymes in the biosynthetic pathway are up-regulated. Western blot analysis and activity measurements showed similar amounts and activities of myrosinase in CYP79A1 and control plants. Thus, the increase in glucosinolate content in CYP79A1 plants was not accompanied by an increase in content or activity of degradation enzyme. The present data demonstrate that the high biosynthetic capacity of the postoxime enzymes combined with a low substrate-specificity of the post-oxime enzymes in A. thaliana provide a highly flexible system for metabolic engineering of glucosinolate profiles, including new (non-endogenous) glucosinolates derived from oximes introduced into the plant, e.g. by transformation with CYP79 homologues.

Comparative genomic hybridization reveals non-random chromosomal aberrations in early preinvasive cervical lesions.

We performed CGH analysis on 34 cervical lesions, which included 8 cases of koilocytosis, 6 mild dysplasias and 20 moderate dysplasias. Chromosome aberrations were detected in 11 cases of which 9 were moderate dysplasias. A total of 55 chromosome arms were involved. The most frequent aberrations were losses of 5p and Xq, each of which was present in 5/34 cases. Gain of 3q was detected in two moderate dysplasias. This aberration is the most frequent copy number change in advanced-stage cervical carcinoma. A considerable number of the aberrations found in the preinvasive cases of this study are frequently present in invasive cervical tumors. The presence of apparently non-random chromosome aberrations in early preinvasive cervical lesions has not previously been described.

Acardiac twin with preserved brain.

The fatal acardiac syndrome is a rare complication of monochorionic twinning and is reported in 1 of 35,000 births. It is caused by arterioarterial and venovenous placental anastomoses leading to circulatory predominance of 1 twin. The donor 'pump' twin provides circulation for itself and for the recipient acardiac twin. The acardiac twin is usually grossly abnormal with severe reduction anomalies of the upper part of the body. We report a twin pregnancy, where a recipient twin initially by ultrasound was misdiagnosed as dead. In the third trimester the supposedly dead twin presented as an edematous acardiac twin without peripheral reduction defects and a nearly normally developed brain. An acardiac twin with a nearly normal external appearance and an almost normally developed brain, nourished by a surviving twin brother, has not previously been described in the literature.

1,4-Dimethoxyglucobrassicin in Barbarea and 4-hydroxyglucobrassicin in Arabidopsis and Brassica.

A novel indole glucosinolate, 1,4-dimethoxyglucobrassicin (1,4-dimethoxyindol-3-ylmethylglucosinolate), was isolated as the desulfo derivative from roots of the P-type of Barbarea vulgaris ssp. arcuata, and its structure was determined by spectroscopy including 2D NMR spectroscopy. 4-Hydroxyglucobrassicin (4-hydroxyindol-3-ylmethylglucosinolate) was isolated as the desulfo derivative from green siliques (fruits) of Arabidopsis thaliana and identified by comparison of its (1)H NMR spectrum with the spectrum of the known desulfoglucosinolate from Brassica napus. The delayed elution of desulfo indole glucosinolates from the DEAE Sephadex column used in sample preparation was examined, and the diode-array UV spectra of desulfo indole glucosinolates were measured, to ensure a reliable determination of 1,4-dimethoxyglucobrassicin and 4-hydroxyglucobrassicin with the existing analysis method based on the HPLC of desulfoglucosinolates. 1,4-Dimethoxyglucobrassicin was not detected in 10 other Arabidopsis, Brassica, and Barbarea species, indicating an evolutionarily recent mutation in the indole glucosinolate biosynthesis in B. vulgaris ssp. arcuata type P.

Influence of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus on Mozzarella cheese and cheese whey.

We investigated the effect of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus starter bacteria on Mozzarella cheese functionality and whey viscosity. Mozzarella cheeses were manufactured with Lactobacillus helveticus LH100 paired with one of four S. thermophilus strains: MR-1C, a bacterium that produces a capsular exopolysaccharide; MTC360, a strain that secretes a ropy exopolysaccharide; TAO61, a nonexopolysaccharide-producing commercial cheese starter; and DM10, a nonencapsulated, exopolysaccharide-negative mutant of strain MR-1C. As expected, cheese moisture levels were significantly higher in Mozzarella cheeses made with exopolysaccharide-positive versus exopolysaccharide-negative streptococci, and melt properties were better in the higher moisture cheeses. Whey viscosity measurements showed that unconcentrated and ultrafiltered, fivefold concentrated whey from cheeses made with S. thermophilus MTC360 were significantly more viscous than whey from cheeses made with MR-1C, TAO61, or DM10. No significant differences were noted between the viscosity of unconcentrated or concentrated whey from cheeses made with S. thermophilus MR-1C versus the industrial cheese starter TAO61. These data indicate that encapsulated, but not ropy, exopolysaccharide-producing S. thermophilus strains can be utilized to increase the moisture level of cheese and to improve the melt properties of Mozzarella cheese without adversely affecting whey viscosity.

Role of parvovirus B19 infection in childhood idiopathic thrombocytopenic purpura.

Although parvovirus B19 exhibits a strong tissue-tropism for erythroid progenitor cells leading to anaemia, several case reports indicate that parvovirus B19 infection may also cause the development of thrombocytopenia. Despite recent studies, the frequency and clinical relevance of this association have remained questionable. Consequently, and in view of the paucity of evidence regarding a viral aetiology for idiopathic thrombocytopenic purpura (ITP), we examined the role of parvovirus B19 in 47 children with newly diagnosed ITP. Specific viral DNA indicating a current or recent parvovirus B19 infection was demonstrated in 6 of 47 patients (13%) employing the polymerase chain reaction technique. Our study suggests that children with ITP and associated parvovirus B19 infection are characterized by acute onset of profound thrombocytopenia. Among the parvovirus B19 positive children, duration of disease was brief in three children treated with immunoglobulin but chronic in the remaining three patients given high-dose steroids. Prospective studies are needed to confirm these initial observations. This virus should be considered as a possible aetiologic agent in some children with ITP.

Distribution of ATPase and ATP-to-ADP phosphate exchange activities in magnesium chelatase subunits of Chlorobium vibrioforme and Synechocystis PCC6803.

Insertion of magnesium into protoporphyrin IX is a complex ATP-dependent reaction catalysed by the enzyme Mg-chelatase. Three separate proteins (Mg-chelatase subunits), designated as D, H and I, are involved in the chelation reaction. The genes encoding the Mg-chelatase subunits of the green sulfur bacterium Chlorobium vibrioforme and of the cyanobacterium Synechocystis strain PCC6803 were expressed in Escherichia coli. The recombinant proteins were purified, tested for ATPase and phosphate exchange activities, and compared with the activities of the corresponding subunits of Rhodobacter sphaeroides. The Synechocystis strain PCC6803 I subunit and the C. vibrioforme H and I subunits hydrolysed ATP at the rates of 2.0, 1.8 and 0.16 nmol (mg protein)-1 min-1, respectively. The ATPase activity of the C. vibrioforme H subunit was similar to that reported for the R. sphaeroides H subunit. The Synechocystis strain PCC6803 H subunit failed to hydrolyse ATP. The I subunit of Synechocystis strain PCC6803 and C. vibrioforme catalysed a transfer of PO4 from ATP to ADP (exchange activity) at the rate of 1.75 +/- 0.15 nmol (mg protein)-1 min-1. This exchange rate was 300-fold lower than that reported for the R. sphaeroides I subunit. The PO4 exchange activities were correlated with the presence of the sequence GXRGTGKSTXVRALA in the primary structure of the three I subunits. Mg-chelatase activity was reconstituted by combining the three subunits of the same bacterium [rates of 41-89 pmol Mg-deuteroporphyrin (mg protein)-1 min-1]. Heterologous subunit combinations resulted in low or no Mg-chelatase activity.