[Molecular classification of bladder cancer. Possible similarities to breast cancer]. / Molekulare Klassifikation des Harnblasenkarzinoms. Mögliche Ähnlichkeit zum Mammakarzinom.

Therapeutic decisions for breast cancer are increasingly becoming based on subtype-specific gene expression tests. For bladder cancer very similar subtypes have been identified by genome-wide mRNA analysis, which as for breast cancer differ with respect to the prognosis and response to therapy on the basis of their hormone dependency. At the DNA level, however, the type of mutations and their frequencies within the subtypes are strikingly different between bladder and breast cancers. It will be interesting to see whether possible driver mutations can serve as therapeutic targets in both indications. In contrast, the apparent hormone dependency of a substantial number of bladder carcinomas suggests that hormonal and anti-hormonal treatment can be valid therapy options similar to breast cancer. Moreover, gender-specific differences with respect to the incidence and aggressiveness of male compared to female bladder cancers can be explained by hormonal effects. Together with forthcoming immunomodulatory therapies these multiple therapy options raise and give new hope to efficiently combat this aggressive disease.

Metabolic intervention on lipid synthesis converging pathways abrogates prostate cancer growth.

One of the most conserved features of all cancers is a profound reprogramming of cellular metabolism, favoring biosynthetic processes and limiting catalytic processes. With the acquired knowledge of some of these important changes, we have designed a combination therapy in order to force cancer cells to use a particular metabolic pathway that ultimately results in the accumulation of toxic products. This innovative approach consists of blocking lipid synthesis, at the same time that we force the cell, through the inhibition of AMP-activated kinase, to accumulate toxic intermediates, such as malonyl-coenzyme A (malonyl-CoA) or nicotinamide adenine dinucleotide phosphate. This results in excess of oxidative stress and cancer cell death. Our new therapeutic strategy, based on the manipulation of metabolic pathways, will certainly set up the basis for new upcoming studies defining a new paradigm of cancer treatment.

Bone-metastatic prostate carcinoma favors mesenchymal stem cell differentiation toward osteoblasts and reduces their osteoclastogenic potential.

Bone homeostasis is achieved by the balance between osteoclast-dependent bone resorption and osteoblastic events involving differentiation of adult mesenchymal stem cells (MSCs). Prostate carcinoma (PC) cells display the propensity to metastasize to bone marrow where they disrupt bone homeostasis as a result of mixed osteolytic and osteoblastic lesions. The PC-dependent activation of osteoclasts represents the initial step of tumor engraftment into bone, followed by an accelerated osteoblastic activity and exaggerated bone formation. However, the interactions between PC cells and MSCs and their participation in the disease progression remain as yet unclear. In this study, we show that bone metastatic PC-3 carcinoma cells release factors that increase the expression by human (h)MSCs of several known pro-osteoblastic commitment factors, such as α5/ß1 integrins, fibronectin, and osteoprotegerin. As a consequence, as shown in an osteogenesis assay, hMSCs treated with conditioned medium (C(ed) M) derived from PC-3 cells have an enhanced potential to differentiate into osteoblasts, as compared to hMSCs treated with control medium or with C(ed) M from non-metastatic 22RV1 cells. We demonstrate that FGF-9, one of the factors produced by PC-3 cells, is involved in this process. Furthermore, we show that PC-3 C(ed) M decreases the pro-osteoclastic activity of hMSCs. Altogether, these findings allow us to propose clues to understand the mechanisms by which PC favors bone synthesis by regulating MSC outcome and properties.

Metabolism and proliferation share common regulatory pathways in cancer cells.

Cancer development involves major alterations in cells' metabolism. Enhanced glycolysis and de novo fatty acids synthesis are indeed characteristic features of cancer. Cell proliferation and metabolism are tightly linked cellular processes. Others and we have previously shown a close relationship between metabolic responses and proliferative stimuli. In addition to trigger proliferative and survival signaling pathways, most oncoproteins also trigger metabolic changes to transform the cell. We present herein the view that participation of cell-cycle regulators and oncogenic proteins to cancer development extend beyond the control of cell proliferation, and discuss how these new functions may be implicated in metabolic alterations concomitant to the pathogenesis of human cancers.

Erythrocyte sedimentation rate as a marker of inflammation and ongoing coagulation in stroke and transient ischaemic attack.

OBJECTIVE: Systemic infection and inflammation have been implicated in the aetiology of thrombotic cerebral events, particularly in younger patients. We decided to determine whether those patients with raised D-dimer levels, indicating continuing thrombosis and fibrinolysis, had evidence of concurrent infection or inflammation as manifested by a raised erythrocyte sedimentation rate (ESR) measured after an ischaemic stroke/transient ischaemic attack (TIA). METHODS: One hundred and forty-eight patients who had suffered either single or recurrent cerebrovascular episodes were analysed. The patients were referred to the thrombosis and haemostasis unit at Johannesburg Hospital for evaluation of their thrombotic profiles, including D-dimer levels. Concurrent infection was assessed by measurement of white cell count (WCC) and ESR. The variable time interval between the date of the most recent cerebrovascular event and the date of venesection was determined. A history was taken, a physical and neurological examination was performed, and a cardiology assessment and neuroimaging studies were done. RESULTS: Raised D-dimer levels correlated significantly with ESR levels (p = 0.0094) in all patients. This was particularly evident when comparing the 70 younger patients (aged less than 45 years) with the 78 older patients (> 45 years) with raised D-dimers (p = 0.0070). When analysing other markers of inflammation/infection in association with raised D-dimer levels and ESR, mean fibrinogen levels were significantly raised at 6.56 g/l (p = 0.0122). An elevated WCC, as a categorical variable, was significantly associated with an elevated ESR (p = 0.0092). CONCLUSION: There is a significant correlation between elevated D-dimer levels (indicating abnormalities of coagulation and fibrinolysis) and markers of inflammatory and/or infective processes. This is particularly evident in black patients below the age of 45 years. These patients are believed to be at decreased risk for generalised atheromatous disease compared with older white patients. The ramifications of these findings are potentially important with regard to thrombotic cerebrovascular disease aetiology, investigation, management and prevention.

The South African stroke risk in general practice study.

BACKGROUND: Incidence of stroke is increasing in sub-Saharan Africa and stroke prevention is an essential component of successful stroke management. General practitioners (GPs) are well placed to manage stroke risk factors. To design appropriate strategies for risk factor reduction we need to know the risk factor prevalence in each of the population groups attending GPs. The aim of this study was to establish the prevalence of stroke risk factors in the South African general practice population. METHOD: We conducted a multicentre, observational study of patients attending general practice in South Africa. Two hundred general practices were randomly selected from lists provided by pharmaceutical representatives. Each GP approached 50 consecutive patients aged 30 years and older. Patients completed an information sheet and the GP documented the patient's risk factors. The resulting sample is relevant if not necessarily representative in a statistical sense. RESULTS: A total of 9 731 questionnaires were returned out of a possible 10,000. The mean age of particpants was 50.7 years. Seventy-six per cent had 1 or more risk factors and 40% had 2 or more risk factors. Hypertension was the commonest risk factor in all population groups (55%) but was highest in black patients (59%). Dyslipidaemia was commonest in whites (37%) and least common in blacks (5%). Diabetes was commonest in Asians (24%) but least common in whites (8%). Risk factors other than smoking increased with age. CONCLUSION: This study provides unique data on the prevalence of stroke risk factors in a South African general practice population. Risk factors are common in all population groups, but differ in distribution among the groups. There is considerable opportunity to reduce the burden of stroke in South Africa through GP screening for and treatment of risk factors.

Sporadic fatal insomnia: a case study.

A 58-year-old man died after a 27-month illness characterized by insomnia, confirmed by polysomnography. He was homozygous for methionine at codon 129 of the prion gene but had no mutation in the prion gene. Neuropathology showed thalamic and olivary atrophy and no spongiform changes. Paraffin-embedded tissue blotting demonstrated abnormal prion protein in the brain. This is the first case of the sporadic form of fatal familial insomnia with demonstration of the disorder by polysomnography.

Bradykinesia akinesia inco-ordination test (BRAIN TEST): an objective computerised assessment of upper limb motor function.

OBJECTIVES: A simple and rapid computerised keyboard test, based on the alternating finger tapping test, has been developed to quantify upper limb motor function. The test generates several variables: (1) kinesia score: the number of keystrokes in 60 seconds; (2) akinesia time: cumulative time that keys are depressed; (3) dysmetria score: a weighted index calculated using the number of incorrectly hit keys corrected for speed; (4) incoordination score: a measure of rhythmicity which corresponds to the variance of the time interval between keystrokes. METHODS: The BRAIN TEST(Copyright ) was assessed on 35 patients with idiopathic Parkinson's disease, 12 patients with cerebellar dysfunction, and 27 normal control subjects. RESULTS: The mean kinesia scores of patients with Parkinson's disease or cerebellar dysfunction were significantly slower than normal controls (Parkinson's disease=107 (SD 28) keys/min v cerebellar dysfunction=86+/- (SD 28) v normal controls=182 (SD 26), p<0.001) and correlated with the UPDRS (r =-0.69, p<0.001). The akinesia time is very insensitive and was only abnormal in patients with severe parkinsonism. The median dysmetria (cerebellar dysfunction=13.8 v Parkinson's disease=6.1 v normal controls=4.2, p=0.002) and inco-ordination scores (cerebellar dysfunction=5.12 v Parkinson's disease=0.84 v normal controls=0.15, p=0.002) were significantly higher in patients with cerebellar dysfunction, in whom the dysmetria score correlated with a cerebellar disease rating scale (r=0.64, p=0.02). CONCLUSION: The BRAIN TEST(Copyright ) provides a simple, rapid, and objective assessment of upper limb motor function. It assesses speed, accuracy, and rhythmicity of upper limb movements regardless of their physiological basis. The results of the test correlate well with clinical rating scales in Parkinson's disease and cerebellar dysfunction. The BRAIN test will be useful in clinical studies. It can be downloaded from the Internet ().

GA2/GM2/GD2 synthase localizes to the trans-golgi network of CHO-K1 cells.

UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as the trans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with d, l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.

Retrograde balloon angioplasty of brachiocephalic or common carotid artery stenoses at the time of carotid endarterectomy.

OBJECTIVES: This study was performed to demonstrate the value and durability of intraoperative retrograde angioplasty for stenotic lesions of the aortic arch branches at the time of carotid endarterectomy for the treatment of tandem proximal and bifurcation carotid lesions. DESIGN: Retrospective analysis of the clinical data. METHODS: Forty-four patients were included in this study when they presented with symptomatic extracranial vascular disease due to stenosis of both a proximal aortic arch branch and carotid bifurcation disease. Tandem disease was detected in the vascular laboratory and confirmed by angiography. Each patient was subjected to conventional carotid endarterectomy, and at the time of operation, the proximal lesion was subjected to transluminal angioplasty through the endarterectomy arteriotomy (brachiocephalic 24; left common carotid 15; right common carotid artery five). Patients were then followed up clinically and by non-invasive tests at 6-monthly intervals. RESULTS: Forty-three successful dilatations were achieved. The single initial technical failure was due to heavy calcification of a brachiocephalic artery. In the follow-up period restenosis was noted in four patients. All restenosis occurred within 24 months. No restenosis at the angioplasty site was noted on subsequent follow-up of the remaining 39 patients. No perioperative stroke or death was encountered. A surprisingly high mortality rate was noted on follow-up in this group of patients, suggesting the presence of more aggressive and advanced diffuse vascular disease. CONCLUSION: Retrograde intraoperative angioplasty of the proximal component of a tandem extracranial lesion has in this series proven to be a safe and durable therapeutic option. This technique has an acceptable restenosis rate in a subset of patients who have been demonstrated to have a shortened life expectancy and a high mortality rate in the follow-up period.

Do stroke patients realise that a consequence of hypertension is stroke?

STUDY OBJECTIVE: The specific objectives of the study were to survey residual disability and handicap following stroke. Information on four risk factors, namely hypertension, age, smoking, and alcohol abuse, was obtained. Enquiry was made into the subjects' insight into the causes of their problems. DESIGN: Descriptive survey. SETTING: Baragwanath Hospital and Soweto. PARTICIPANTS: Stroke patients 12-14 weeks post-discharge. OUTCOME MEASURES: Structured questionnaire. RESULTS: A total of 361 patients were initially screened. Only 54 fulfilled all inclusion criteria, 38 (70%) over 50 years of age and 16 (30%) under 50 years. Ninety-three of the 361 died within the first 3 months; 71% of all patients knew that they had suffered a stroke. Only 20% of the total group understood that hypertension had probably caused their stroke, although 76% of the older group and 56% of the younger group had been told at some stage that they were hypertensive. Of the older group 32% knew the name of their medication, 21% could not name their medication and 23% claimed they were on no medication. Similarly in the younger group, 19% could name their medication, 25% could not name their medication, and 12% were on no medication. In addition 16% of the older group and 56% of the younger group admitted to smoking. The abuse of alcohol in both groups was low, but this figure was taken from subjective assessment and may not reflect the true extent of drinking as a risk factor. CONCLUSION: Most patients in this study appear well aware of their hypertension and take medication. However, they seem unaware that their hypertension and stroke are causally linked and their hypertension knowledge is suboptimal. It is also apparent that smoking is increasing as a major risk factor for stroke in the black population of South Africa. Patients need more education regarding hypertension and its consequences.

Aphanomyces euteiches Inoculum Potential: A Rolled-Towel Bioassay Suitable for Fine-Textured Soils.

A sensitive measure of soil inoculum potential is needed to evaluate field management of common root rot (Aphanomyces euteiches) in peas (Pisum sativum). A modified rolled-towel (RT) bioassay had been proposed to measure soil inoculum potential in fine-textured soils used for pea production in Minnesota. Homogenized soil was used because organic debris containing the inoculum could not be separated by wet sieving. The poor precision prompted an evaluation of procedures to improve this modified RT bioassay. Seed treatment with a 5% solution of sodium hypochlorite before pea seed germination and plant isolation procedures during the RT bioassay preparation/incubation reduced seedborne contamination and seedling loss to less than 5%. Tests conducted with pasteurized soil that was artificially infested with oospores showed the region of the pea taproot 1 to 2 cm below the seed to be more susceptible to infection (33% compared with 15% infected seedlings) than the region 1 to 2 cm above the root tip. A soil volume of 1.0 cm3 increased inoculum potential compared with 0.5 cm3 applied to each seedling but did not influence the random error; the 40-seedling compared with the 20-seedling RT bioassay reduced random error from 18 to 12%. The modified RT bioassay conducted on soil that was artificially infested after steam treatment or without steam treatment showed superior performance when using 40 seedlings compared with 20 seedlings when evaluated for accuracy and precision. Multiple infection theory demonstrated more multiple infections in the RT bioassay with a 0.5 cm3 soil volume applied to each seedling, which shows that soil mass is a factor preventing a higher percentage of infected seedlings. These modifications to the RT bioassay improved the method enough to reduce the random error by one-half compared with using homogenized soil without the proposed modifications.

Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase.

GM3-positive Chinese hamster ovary cells (CHO-K1 cells) lack the ability to synthesize GM2 and the complex gangliosides GM1 and GD1a from [3H]Gal added to the culture medium. However, they acquire the ability to synthesize GM2 and to synthesize and immunoexpress complex gangliosides upon transient transfection with a cDNA encoding the human GM3:N-acetylgalactosaminyl transferase (GM2 synthase). The activities of endogenous GM1- and GD1a-synthases in the parental cell line and in cells transfected with the plasmid with or without the GM2 synthase cDNA were essentially identical and comparable in terms of specific activity with the endogenous GM3 synthase. Results indicate that glycosyltransferases acting on GM2 to produce GM1 and GD1a are constitutively present in CHO-K1 cells, and that the expression of their activities depend on the supply of the acceptor GM2. In addition, these results lend support to the notion that GM2 synthase is a key regulatory enzyme influencing the balance between simple and complex gangliosides.

Cloning, characterization and developmental expression of alpha2,8 sialyltransferase (GD3 synthase, ST8Sia I) gene in chick brain and retina.

GD3 and GM2 synthases act on ganglioside GM3 at the branching point of the pathway of synthesis of gangliosides in which the "a", "b" and "c" families are produced. The relative activities of these enzymes are important for regulating the ganglioside composition of a given tissue. In the present work, we report the cloning and characterization of a chick GD3 synthase cDNA. The cloned cDNA directed the synthesis of a functionally active enzyme in transiently transfected CHO-K1 cells and was highly homologous to mammalian GD3 synthases. In Northern blot experiments the cDNA detected a single specific GD3 synthase mRNA of about 9.0 kb both in the chicken brain and retina. The abundance of the specific mRNA transcript declined steadily from E7-E9 to very low values around PN2. The levels of enzyme activities measured at the same developmental stages roughly followed the changes of specific mRNA levels in both tissues. In situ hybridization of embryonic neural retina cells in culture showed that both glial- and neuron-like cells expressed the specific GD3 synthase mRNA, although with different intensities. Results indicate that transcription and/or stability of the specific GD3 synthase mRNA constitute a level of control of the expression of GD3 synthase and indirectly of the ganglioside composition in the developing chicken central nervous system (CNS).

Expression of beta 1-4 N-acetylgalactosaminyltransferase gene in the developing rat brain and retina: mRNA, protein immunoreactivity and enzyme activity.

The developmental pattern of expression of the UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) gene was examined in the rat brain and retina. A GalNAc-T cDNA cloned from a rat olfactory bulb cDNA library was used as a probe for Northern blot and in situ hybridization experiments and a rabbit polyclonal antibody to rat GalNAc-T peptide was used for Western blot analysis. In Northern blot experiments, a single approximately 3 kb transcript was detected both in brain and retina. In brain, the abundance of this transcript increased from E15 to PN1-5 and then declined while, in retina, it increased steadily from PN1 to PN13-24. The developmental trends of GalNAc-T mRNA expression, GalNAc-T immunoreactive protein and GalNAc-T activity were comparable in brain. In retina, however, GalNAc-T activity and GalNAc-T peptide immunoreactivity followed developmental patterns that were similar between them and different from that of the specific mRNA. Results suggest that post-transcriptional controls of the GalNAc-T gene expression operate in the rat CNS, which are particularly evident in retina. The expression of the GalNAc-T gene in glial and neuronal cells was examined in rat retina cell cultures by in situ hybridization. The GalNAc-T mRNA was abundant in GM1+/GD3+ neurons and almost absent in the flat, GM1-/GD3+ Müller glia-derived cells.

Myxoma in the carotid artery. Myxomatous occlusion of internal carotid artery.

BACKGROUND: We describe a patient with an unusual cause of internal carotid artery occlusion resulting in a stroke. CASE DESCRIPTION: A 41-year-old woman presented with a typical acute right middle cerebral artery territory infarct. Her hematological and cardiological status was assessed, including all extracranial vessels. Carotid angiography and a biopsy were performed of the occluded right internal carotid artery and demonstrated a myxoma. Cardiac investigations to determine the source of the myxoma, including transthoracic and transesophageal echocardiograms, CT and yo-yo CT scans, and MRI of the heart, were normal. No residual tumor or potential source of the tumor was found. CONCLUSION: The cause of stroke was a myxomatous occlusion of the right internal carotid artery. An entire cardiac tumor may have embolized with no detectable residual tumor in the heart; alternatively, a myxoma may have originated as a primary tumor in the carotid artery. To our knowledge, no primary myxoma has been reported to have originated in a blood vessel.

GT3 synthesis in the proximal Golgi occurs in a compartment different from those for GD3 and GM3 synthesis.

Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [3H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[3H]Gal. In (a), 1 microM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to approximately 50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of > 90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited approximately 30 and 70%, respectively, in 7- and 10-day cells. In (b), > 80% of the [3H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [3H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, < 5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[3H]NeuAc incorporated approximately 20% of [3H]NeuAc into endogenous GT3, and this percentage was not affected by 1 microM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.