Synaptic dysfunction, memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons.

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.

The mast cells of the human uterus.

Staining of mast cells in the human uterus has been studied using four fixatives and five staining methods to determine whether there are subpopulations of mucosal (endometrial) and connective tissue (myometrial) mast cells, and to discover how they can best be demonstrated. Following formalin fixation none of the staining methods showed maximum staining of mast cells in either endometrium or myometrium. The best demonstration of uterine mast cells is by fixation with either isotonic formol acetic acid or Mota's basic lead acetate followed by staining with the long toluidine blue technique. Although the degree of MC understaining following formalin fixation was greater for the endometrium than for the myometrium this is inadequate evidence to designate two cell populations. The findings suggest that the mast cells of the human uterus are all one population but show heterogeneity of histological properties possibly related to their functional state.

Immunohistochemical study of vascular injury in acute multiple sclerosis.

AIMS: To examine the vascular changes occurring in three archival cases of acute multiple sclerosis, and to provide immunohistochemical evidence of early endothelial cell activation and vascular occlusion in this condition. METHODS: Central nervous system tissues from three cases of acute active multiple sclerosis and six non-inflammatory controls were stained using the following methods: haematoxylin and eosin, Luxol fast blue, cresyl violet, Bielschowsky's silver, and reticulin. Tissues were also immunostained with specific antibodies against collagen type IV, factor XIIIa, class II antigens, glial fibrillary acidic protein, and fibrinogen. RESULTS: Early vascular endothelial cell activation which may progress to vasculitis and vascular occlusion including class II antigen expression and fibrin deposition were identified. The vascular changes were seen prior to cerebral parenchymal reaction and demyelination, and were not seen in control cerebral tissues. CONCLUSION: It is proposed that vascular endothelial cell activation may be an early and pivotal event in the evolution of multiple sclerosis, and that demyelination may have an ischaemic basis in this condition. The vascular endothelium may contain an early element in the evolution of multiple sclerosis.

Immunohistochemical study of tissue factor expression in normal intestine and idiopathic inflammatory bowel disease.

AIMS: To investigate the localisation of tissue factor expression in normal and inflamed intestine. METHODS: Serial cryostat sections of tissue taken from patients with Crohn's disease (n = 8), ulcerative colitis (n = 5), and from controls (n = 5) were stained with haematoxylin and eosin and immunostained for tissue factor, collagen type IV, fibrinogen and platelet glycoprotein IIIa. RESULTS: In control tissues tissue factor was present as a continuous layer along the epithelial basal lamina: sections from controls did not immunostain for fibrinogen or platelets. In non-ulcerated inflamed mucosa, tissue factor staining intensified in cases of Crohn's disease and was associated with fibrin deposition. Staining for tissue factor was either patchy or absent in cases of ulcerative colitis and there was no fibrin deposition. This change accompanied the early destruction of the epithelial basal lamina in ulcerative colitis that was not seen in Crohn's disease. In both diseases tissue factor expression in severely inflamed and ulcerated mucosa was present on lamina propria macrophages and vascular endothelium and was associated with fibrin or platelet thrombi. In three of eight cases of Crohn's disease tissue factor expression and thrombi were evident in areas of submucosal vasculitis. These were not seen in adjacent normal vessels. CONCLUSIONS: These observations are consistent with a tissue factor haemostatic barrier in the intestine: this barrier seems to be incomplete or defective in ulcerative colitis. Tissue factor expression by macrophages and endothelial cells may be important, particularly in the microvascular thrombosis and induration which are characteristic of Crohn's disease.

Pulmonary platelet aggregates: possible cause of sudden peroperative death in adults undergoing liver transplantation.

AIMS: To determine if massive pulmonary platelet thromboembolism is a common cause of peroperative death following liver transplantation; and to compare the incidence of this event with patients dying after non-transplantation procedures. METHODS: Necropsy tissues from all patients dying within 10 days of operation during the past three and a half years were studied (six liver transplantations and 13 unrelated operations). Haematoxylin and eosin stained sections of all tissues were examined. Additional sections of lung tissue were immunostained for constituents of thrombus (fibrin and platelets). RESULTS: At necropsy the lungs from all six liver transplant recipients were heavy with a rubbery texture and little oedema fluid. Those from non-transplantation patients appeared normal or very oedematous. Microscopic examination showed that there were numerous platelet aggregates occluding pulmonary capillaries in all six transplant recipients, but in only three of the non-transplant patients. These thrombi were numerous in patients dying during surgery and the number was underestimated in routine sections because of the surrounding capillary congestion. Detection was improved by immunostaining for platelets with factor XIIIA and platelet glyco-protein IIIa. CONCLUSIONS: Massive platelet thromboembolism is a likely cause of death in patients dying unexpectedly following recent liver transplantation. Non-transplantation patients dying during surgery who show similar appearances usually have conditions known to have a high risk of thrombosis or embolism (cement hypotension syndrome and disseminated intravascular coagulation). The cause of this extensive platelet activation in liver transplant recipients is uncertain and may be multifactorial. The unusual rubbery consistency of the lungs on macroscopic examination could alert the pathologist to the underlying condition. Immunostaining for platelets improves the detection microscopically.

Forward mutation of S. typhimurium by smokeless tobacco extracts.

Mutagenicity of 4 popular brands of smokeless tobaccos was studied using a S. typhimurium forward mutation assay. Aqueous extracts of 4 brands and dichloromethane and methanol extracts of 1 of the 4 brands of smokeless tobacco's did not induce significant mutagenicity either in the presence or absence of metabolic activation. Aqueous and organic extracts were however mutagenic when treated with physiological levels of sodium nitrite (0.25 mM) at acidic pH and without metabolic activation. The results indicate that smokeless tobacco contain polar and non-polar chemicals which become mutagenic to S. typhimurium under nitrosation conditions.

Long-term low-dose mutation studies in human cells: metanil yellow and orange II.

We tested the mutagenic effects of two commonly used fold colors, metanil yellow and orange II, in AHH-1 human lymphoblast cells. The cell line, which is competent for oxidative metabolism of various chemicals, was exposed to both compounds in high-dose x short-term (3 day) or high-dose x long-term (10-day) and low-dose x long-term (20-day) treatments. Concentrations of metanil yellow and orange II as low as 22 nM and 12 nM, respectively, were sufficient to induce mutation rates which were equal to twice the spontaneous mutation rate at the HPRT locus in AHH-1 cells.

Genetic effects of 5-hydroxymethyl-2'-deoxyuridine, a product of ionizing radiation.

Ionizing radiation causes formation of heterogeneous types of damage to DNA. Among those, 5-hydroxymethyl-2'-deoxyuridine (HMdU) was identified as a major thymidine derivative in gamma-irradiated HeLa cells [G.W. Teebor, K. Frenkel and M.S. Goldstein (1984) Proc. Natl. Acad. Sci. (U.S.A.), 81, 318-321]. We report here that HMdU is a strong inducer of lambda prophage in Escherichia coli WP2s(lambda) and is highy mutagenic in Salmonella typhimurium. HMdU causes his+ revertants in strains TA100, which reverts predominantly by base-pair substitution at G-C sites, and TA97, which reverts mainly by frameshift mutation at G-C sites. It does not cause reversion in TA98, another frameshift-sensitive strain, nor in strains TA1535 and TA1537. Of those tested, only the last two strains do not contain pkM101, a plasmid which enhances mutagenic effects of ionizing radiation. HMdU also causes reversion in strains TA102 and TA104, which detect oxidative damage and can revert by base-pair substitution at A-T base pairs at the hisG428 site. We show that HMdU can be incorporated into DNA of TA100 and that, in addition to causing point mutations, it causes suppressor mutations as well. The ability of HMdU to induce lambda prophage and its strong mutagenicity in Salmonella typhimurium provide evidence that the presence of HMdU in DNA is biologically significant and may play a major role in the genetic consequences of ionizing radiation and other types of oxidative damage.

Liver death and regeneration in paracetamol toxicity.

Paracetamol overdose (POD) is a major clinical problem as the commonest cause of fulminant hepatic failure (FHF) in the UK and the USA. While the main loss of liver mass occurs following hepatocyte necrosis, hepatocyte apoptosis has also been reported to occur during paracetamol toxicity in murine liver. Hepatocyte apoptosis has not previously been identified in human liver and the significance of apoptosis in paracetamol toxicity is not known. In this study of paracetamol toxicity in human liver after POD, hepatocyte apoptosis was identified at time of liver transplantation or death and was associated with striking regenerative activity. The biological significance of apoptosis is unclear but the rates of apoptosis found (0.6%) could account for a significant loss of hepatic parenchyma. The stimulus for apoptosis is not known but it is unlikely to be induced directly by paracetamol since it is absent from serum at this time. The possibility that apoptosis may be induced by Kupffer cell activation with cytokine production is raised. Patients who develop FHF after POD have a poor prognosis, with few therapeutic options apart from liver transplantation; an understanding of the dynamics of liver regeneration and ongoing cell loss by apoptosis may allow the development of new therapies in these patients.

Drawing specimens for coagulation testing: is a second tube necessary?

Three recent studies discussed the possibility that the National Committee for Clinical Laboratory Standards (NCCLS) recommendations that the coagulation specimen should be the second or third tube collected are unnecessary. However, only one reagent/instrument was used in each study. Our protocol differed from the previous studies because we performed the assays on three different reagent/instrument systems on the same samples. Our study used photo-optic, mechanical, and nephelometric systems of clot detection. After obtaining informed consent, we obtained two blue-stoppered tubes of blood from 95 subjects: 15 normal patients and 80 patients currently on coumadin therapy. No discard tube was drawn for coagulation testing. A prothrombin time with an international normalized ratio and an activated partial thromboplastin time, were performed on each tube. Laboratory One used a MLA 1600C (Hemoliance) with Thromboplastin DS (Pacific-Hemostasis, ISI of 1.11) and APTT-LS (Pacific-Hemostasis). Laboratory Two used an STA (Diagnostica-Stago) with Neoplastine CI+ (Diagnostica-Stago, ISI of 1.14) and PTT-LT (Diagnostica-Stago). Laboratory Three used an ACL 300 with Plastinex (Biodata, ISI of 1.67) and Actin FSL (Dade Behring). No clinical or statistically significant differences were seen between the first or second tubes on any of the three reagent/instrument combinations in the PT in seconds, international normalized ratio reporting, or APTT results. Our results indicate that the NCCLS guidelines for obtaining a second tube when performing coagulation testing should be considered for elimination when new revisions are published.

Normal concentrations of heavy metals in autistic spectrum disorders.

AIM: Autism is a neurological-psychiatric disease. In the last 20 years we witnessed a strong increase of autism diagnoses. To explain this increase, some scientists put forward the hypothesis that heavy metal intoxication may be one of the causes of autism. The origin of such an intoxication was hypothesised to be vaccines containing thimerosal as antimicrobic preservative. This preservative is mainly made up of mercury. The aim of our research was to investigate the correlation between autism and high biological concentrations of heavy metals. METHODS: Seventeen autistic patients, between 6 and 16 years old (average: 11.52 DS: 3.20) (15 males and 2 females), were investigated, as well as 20 non autistic subjects from neuropsychiatric service between 6 and 16 years (average: 10.41 DS: 3.20) (15 males and 2 females). In both groups blood, urine and hair samples were analysed trough means of a semiquantitative analysis of heavy metal dosing. The metals analysed were Lead, mercury, cadmium and aluminium, since their build-up may give both neurological and psychiatric symptoms. RESULTS: The comparison of the mean values of the concentrations between the groups, performed with ANOVA test, has shown no statistically relevant differences. CONCLUSION: There wasn't correlation between autism and heavy metal concentration.

An algorithm for the treatment of type 2 diabetes in Latin America.

Diabetes is a principal and growing health concern in Latin America, accounting for significant mortality and morbidities. Large, randomized, prospective trials of various interventional therapies in patients with both type 1 and type 2 diabetes have demonstrated that reductions in hyperglycaemia and management of diabetes-related risk factors can significantly reduce the micro- and macrovascular complications of diabetes. Therefore, patients with type 2 diabetes will benefit from more aggressive treatment regimens to help decrease the occurrence and rate of progression of diabetic complications. Given the many complexities of diabetes management, it is often difficult for general practice physicians to stay abreast of emerging treatment strategies and therapies. Owing to the high prevalence of type 2 diabetes in Latin America, the majority of patients with diabetes are treated by generalists rather than specialists. This article was intended to assist physicians and other healthcare professionals in developing and using effective treatment strategies to stem the growing epidemic of diabetes and its complications in Latin America.

Structure elucidation of a flavonoid glycoside from the roots of Clerodendrum serratum (L.) Moon, Lamiaceae

Apigenin-7-glucoside, C21H20O10 (7-(β-D-glucopyranosyloxy)-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), was first time isolated from the roots of Clerodendrum serratum (L.) Moon, Lamiaceae. Structure elucidation of the compound was carried out by ¹H NMR and FAB-MS studies.

Apigenin-7-glucosídeo, C21H20O10 (7-(β-D-glucopiranosiloxi)-5-hidroxi-2-(4-hidroxifenil)-4H-1-benzopiran-4-ona), foi isolado pela primeira vez das raízes de Clerodendrum serratum (L.) Moon, Lamiaceae. A elucidação estrutural da susbtância foi feita através de estudos de ¹H NMR e FAB-MS.

Smokeless tobacco extracts mutate human cells.

Two commercial brands of smokeless tobacco were extracted with water and these extracts were tested in human cell mutation assays. Using the human cell line TK-6 which expresses no cytochrome P450, the two extracts tested were found to be detectably mutagenic in the range 1-3 mg/ml extractable solids. In AHH-1 cells which constitutively express cytochrome P450IAI, a similar result was found for both brands tested. The two extracts were treated with neutral nitrite solutions to mimic physiologic oral conditions or acidic conditions or acidic conditions with nitrite to mimic physiologic gastric conditions. The mutagenicity of both extracts for both TK-6 and AHH-1 cells was markedly decreased by treatment at neutral pH with sodium nitrite (0.25 mM) and by acidic treatment (2 h, pH 3.0). Treatment of extracts with sodium nitrite at pH 3.0 did not have any effect on the mutagenicity of the untreated extracts for TK-6 cells. The mutagenicity of both the extracts destroyed by acidic treatment however, seemed to be restored to a level equivalent to the mutagenicity of the untreated extracts for the TK-6 cells. The same series of experiments with P450-proficient AHH-1 cells showed uniform reduction of mutagenic activity. Since the two cell lines are equally sensitive to mutation by aqueous tobacco extracts it is concluded that mutagenicity is not cytochrome P450 mediated. It would further appear that the extract mutagen(s) is acid and neutral nitrite labile.

QBEnd/10: a new immunostain for the routine diagnosis of Kaposi's sarcoma.

Kaposi's sarcomas are seen more commonly in routine histopathology laboratories since the advent of the more widespread and aggressive variant of the disease associated with HIV infection. Distinguishing nodular lesions from other spindle cell and vascular tumours can sometimes be difficult. Immunohistochemistry has been disappointing as a diagnostic aid, often requiring special fixation or frozen tissue and even then, staining of spindle cells has been variable. We describe the use of the new IgG1 mouse monoclonal antibody raised against human placental endothelial cells, QBEnd/10, on routine formalin-fixed, paraffin-embedded tissue. A retrospective study was performed on 22 Kaposi's sarcomas of skin including patch, plaque, and nodular lesions and compared with 38 other vascular and spindle cell tumours from skin. All sections were stained with haematoxylin and eosin, QBEnd/10, Ulex europaeus agglutinin 1 (UEA-1) and for factor VIII-related antigen (FVIIIRAg). The results demonstrate that spindle cells in lesions from Kaposi's sarcomas, but not other vascular or spindle cell tumours, immunostain clearly with QBEnd/10. Immunostaining for FVIIIRAg shows only weak and irregular positivity of the spindle cells, whilst staining with UEA-1 is consistently negative. We find that immunostaining with QBEnd/10 aids the diagnosis of Kaposi's sarcomas and allows their distinction from other spindle cell neoplasms of skin in routinely processed material.

Hepatobiliary cystadenoma with mesenchymal stroma: expression of oestrogen receptors in formalin-fixed tissue.

We report three cases of hepatobiliary cystadenoma with mesenchymal stroma in which oestrogen receptor immunostaining was carried out, after using a microwave method of antigen retrieval. In one of the tumours immunoreactivity for oestrogen receptors was demonstrated within the mesenchymal stromal cells. The presence of oestrogen receptors supports the theory that oestrogens act as tumour promoters and may explain the female predilection.

Mast cells in the female genital tract.

Mast cells in the human uterus and adnexa have been studied using basic lead acetate fixation and a long toluidine-blue technique to maximise the numbers of cells stained. Counts were performed on measured areas of tissue and the numbers of mast cells related to clinical and pathologic variables. Considerable variation in numbers was found among individual cases at all the sites studied. In the endometrium and myometrium, a drop in the number of mast cells has been demonstrated with advancing age, particularly after menopause. In leiomyomas the highest counts were in the smaller and more cellular lesions. It is concluded that the numbers of mast cells are at least partly related to the degree of cellularity or atrophy of the surrounding tissues. No significant association was found with menorrhagia or with the presence of leiomyomas.

Neuroblastoma: changing incidence and survival in young people aged 0-24 years. A report from the North of England Young Persons' Malignant Disease Registry.

BACKGROUND AND PROCEDURE: Population based data for neuroblastoma in children and young adults under 25 years at diagnosis were ascertained from the Northern Region Young Persons' Malignant Disease Registry for the period 1968-1995. Age-standardised incidence rates were calculated (ASR) and changes in incidence and survival were investigated. Over the study period 144 patients were registered, of these 136 were children under 15 years at diagnosis (median age: 2.2 years, ASR: 8.6 cases per million children per year), and 8 were 15-24 years (ASR 0.6). RESULTS AND CONCLUSIONS: Incidence of childhood neuroblastoma in the North of England increased significantly over time; ASRs were 5.8 for 1968-1981 and 9.5 for 1982-1995 (rate ratio: 1.6, 95%; CI 1.2-2.3). The increase in incidence was seen in both infants and older children, and in both low stage and advanced disease. Overall 5 year survival was 15% for 1968-1981 and 40% for 1982-1995 (P < 0.0001). Significant improvements in survival were documented across different stage and age-groups, including those over 1 with stage 4 disease (0% versus 18%, P < 0.0001). Further research is needed to investigate the reasons for the increasing incidence of neuroblastoma.

A survey of the nutritional and haemagglutination properties of legume seeds generally available in the UK.

Eighty-five samples from fifteen different legume seed lines generally available in the UK were examined by measurements of their net protein utilization by rats and by haemagglutination tests with erythrocytes from a number of different animal species. From these results the seeds were classified into four broad groups. Group a seeds from most varieties of kidney (Phaseolus vulgaris), runner (Phaseolus coccineus) and tepary (Phaseolus acutifolius) beans showed high reactivity with all cell types and were also highly toxic. Group b, which contained seeds from lima or butter beans (Phaseolus lunatus) and winged bean (Psophocarpus tetragonolobus), agglutinated only human and pronase-treated rat erythrocytes. These seeds did not support proper growth of the rats although the animals survived the 10 d experimental period. Group c consisted of seeds from lentils (Lens culinaris), peas (Pisum sativum), chick-peas (Cicer arietinum), blackeyed peas (Vigna sinensis), pigeon peas (Cajanus cajan), mung beans (Phaseolus aureus), field or broad beans (Vicia faba) and aduki beans (Phaseolus angularis). These generally had low reactivity with all cells and were non-toxic. Group d, represented by soya (Glycine max) and pinto (Phaseolus vulgaris) beans, generally had low reactivity with all cells but caused growth depression at certain dietary concentrations. This growth depression was probably mainly due to antinutritional factors other than lectins. Lectins from group a seeds showed many structural and immunological similarities. However the subunit composition of the lectin from the tepary bean samples was different from that of the other bean lectins in this or any other groups.