No evidence for an ischemic penumbra in massive experimental intracerebral hemorrhage

OBJECTIVES: To determine the effect of massive intracerebral hemorrhage (ICH) on regional cerebral blood flow (rCBF) and metabolism, and to test the hypothesis that there is persistent ischemia in the perihematoma region after ICH. BACKGROUND: Cerebral ischemia is postulated to be one of the mechanisms of neural injury after ICH. Presumably the hematoma induces ischemia by mechanical compression of the surrounding microvasculature. METHODS: The authors induced ICH in eight anesthetized mongrel dogs by autologous blood injection (7.5 mL) under arterial pressure in the deep white matter adjacent to the left basal ganglia. They measured serial rCBF using radiolabeled microspheres in regions around and distant to the hematoma, as well as cerebral oxygen extraction, oxygen consumption (CMRO2), glucose utilization, and lactate production by serial sampling of cerebral venous blood from the sagittal sinus. Mean arterial pressure (MAP) and intracranial pressure (ICP) were monitored continuously. All measurements were recorded at 0.5, 1.0, 2.0, 3.5, and 5.0 hours after induction of ICH and compared with prehematoma values. Evans Blue dye was injected at the end of the experiment, and intensity of staining was compared with three control animals. RESULTS: Compared with prehematoma ICP (12.5+/-2.0 mm Hg, mean+/-standard error), significant elevation in ICP was observed after ICH peaking at 5 hours (34.4+/-5.2 mm Hg). Compared with prehematoma MAP (125.8+/-7.0 mm Hg), significant elevation in MAP was observed at 120 minutes after onset of hematoma (139.1+/-4.6 mm Hg), with return to the prehematoma value by 5 hours. There were no significant changes observed in cerebral oxygen extraction (51.4+/-4.3% versus 44.8+/-4.9%) and CMRO2 (1.8+/-0.3 versus 1.64+/-0.2 mL O2/100 g/min) at 5 hours posthematoma (or any other posthematoma measurement) compared with prehematoma values. There were no significant differences observed in rCBF in the perihematoma gray (18.2+/-0.9 mL/100 g/min versus 20.1+/-1.5 mL/100 g/min) or white matter (15.6+/-1.4 mL/100 g/min versus 15.3+/-1.1 mL/100 g/min) at 5 hours posthematoma (or any other posthematoma measurement) compared with prehematoma values. No changes were observed in cerebral glucose utilization, lactate production, and rCBF in other regions after introduction of ICH. Permeability of the blood-brain barrier was more prominent in the ipsilateral hemisphere in animals with ICH compared with control animals. CONCLUSIONS: Despite a prominent increase in ICP and MAP after ICH, the authors found no evidence to support the presence of an ischemic penumbra in the first 5 hours after ICH. Thus, other mechanisms for acute neural injury and late rCBF changes after ICH must be investigated.     

A role for Edman degradation in proteome studies

Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottleneck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.     

Depression in Parkinson's disease is not accompanied by more corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus

BACKGROUND: Depression is frequently encountered in Parkinson's disease (PD). In addition, more than half of the PD patients have a disturbed dexamethasone suppression test, which is associated with increased activity of corticotropin-releasing hormone (CRH) neurons. We recently found an increase in CRH neuron number, CRH-messenger RNA, and vasopressin colocalization in CRH neurons in the paraventricular nucleus (PVN) of depressed patients, which may be involved in the pathogenesis of depression. METHODS: The number of neurons expressing CRH was determined in the PVN of 6 depressed PD patients with a high score (> or = 13) on the Hamilton Depression Rating Scale, 6 nondepressed PD patients, and 6 controls. RESULTS: The three groups did not differ in the number of neurons expressing CRH. CONCLUSIONS: We hypothesize that activation of CRH neurons in the PVN, as we recently observed in idiopathic depression, does not play an essential role in depression in PD.     

Immunoglobulin A levels in southern elephant seal (Mirounga leonina) milk during the suckling period

Immunoglobulin A (IgA) levels in milk samples from southern elephant seals at King George Island, Antarctica are reported. IgA levels were determined throughout the suckling period (approximately 23 days). The IgA concentration in southern elephant seal milk was lower than in other mammals and, unlike most mammalian milk, was not high during early lactation. There was not a definite pattern in IgA levels, which fluctuated within narrow limits throughout the suckling period (mean +/- SD, 30.81 +/- 6.38 mg IgA/100 g milk). If IgG was present, its level was too low to be detected by the method used. This is the first evidence in Southern elephant seal of the possibility of transmission of passive immunity after birth involving secretion of IgA in the milk.     

Aggravated decrease in the activity of nucleus basalis neurons in Alzheimer's disease is apolipoprotein E-type dependent

As reported before, the metabolic activity of nucleus basalis neurons is reduced significantly in Alzheimer patients. Because the apolipoprotein E (ApoE) epsilon4 genotype is a major risk factor for Alzheimer's disease (AD), we determined whether the decrease in metabolic activity in nucleus basalis neurons in AD is ApoE-type dependent. The size of the Golgi apparatus (GA) was determined as a measure of neuronal metabolic activity in 30 controls and 41 AD patients with a known ApoE genotype by using an image analysis system in the nucleus basalis of Meynert. A polyclonal antibody directed against MG-160, a sialoglycoprotein of the GA, was used to visualize this organelle. There was a very strong reduction in the size of the GA in the nucleus basalis of AD patients. Furthermore, a strong and significant extra reduction in the size of the GA was found in the nucleus basalis neurons of AD patients with either one or two ApoE epsilon4 alleles compared with Alzheimer patients without ApoE epsilon4 alleles. Our data show that the decreased activity of nucleus basalis neurons in AD is ApoE epsilon4 dependent and suggest that ApoE epsilon4 participates in the pathogenesis of AD by decreasing neuronal metabolism.     

CBF beta-SMMHC, expressed in M4Eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells

CBF beta-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBF beta-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBF beta-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBF beta-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBF beta. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBF beta-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBF beta-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBF beta-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor alpha or beta subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBF beta-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBF beta-SMMHC by allowing its increased expression.     

DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine

The methyltransferase of the EcoK type I restriction/modification system is trimeric, M2S1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor S-adenosyl-L-methionine. Our results indicate that the methyltransferase has two non-interacting S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3.60 (+/- 0.42) microM determined by equilibrium dialysis, or 2.21 (+/- 0.29) microM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that S-adenosyl-L-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to S-adenosyl-L-methionine binding. Gel retardation results show that the binding of S-adenosyl-L-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.     

Dietary energy restriction does not inhibit pancreatic carcinogenesis by N-nitrosobis-2-(oxopropyl)amine in the Syrian hamster

Dietary energy restriction was previously shown to be effective in preventing a wide range of experimentally induced cancers. Studies were conducted to assess the influence on pancreatic carcinogenesis of dietary energy restrictions (reduced fat and carbohydrate) of 10%, 20% or 40% in comparison with control in Syrian hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Two carcinogenesis studies were conducted. One used a single treatment with 20 mg BOP/kg body weight and followed hamsters for 102 weeks following treatment, and the other used three weekly treatments of 20 mg BOP/kg body weight and followed hamsters for 45 weeks after treatment. Hamsters were fed control or energy restricted diet beginning the week following the last BOP treatment. Pancreatic carcinomas were induced in 9-18% of the hamsters in the first experiment and in 59-66% of the animals in the second. Dietary energy restriction did not influence carcinoma incidence in either study, and in the second experiment the multiplicity of tumors was higher in the 40% energy restriction (ER) group than in control hamsters. Plasma corticosterone was suppressed by BOP treatment, particularly in the 20% and 40% ER hamsters in the second experiment, and diet or BOP treatment did not significantly alter plasma cortisol. Pancreatic protein kinase Czeta measured by Western blot was highest in the cytosol and particulate fractions of the 40% ER hamsters in the first experiment. These results indicate that dietary energy restriction is not effective in the prevention of BOP induced pancreatic carcinogenesis in the Syrian hamster.