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Biomedical subjects

G S Robertson

Publications and source records attributed to G S Robertson.

At least 19 recordsLinked to original sources
PubMed1999

Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus.

Transient forebrain ischemia produced by four-vessel occlusion (4-VO) triggers the delayed death of CA1 neurons in the hippocampus, resulting in behavioral deficits of spatial learning performance. We demonstrate that CA1 neuronal loss induced by 4-VO (12 min) is preceded by a selective and marked elevation of catalytically active caspase-3 in these neurons, indicative of apoptosis. Virally mediated overexpression of the anti-apoptotic gene X chromosome-linked inhibitor of apoptosis protein (XIAP) prevented both the production of catalytically active caspase-3 and degeneration of CA1 neurons after transient forebrain ischemia. CA1 neurons protected in this manner appeared to function normally, as assessed by immunohistochemical detection of the neuronal activity marker nerve growth factor inducible-A and by spatial learning performance in the Morris water maze. These findings indicate that caspase-3 activation is a key event in ischemic neuronal death and that blockade of this event by XIAP overexpression permits CA1 neurons to survive and operate properly after an ischemic insult.

Animals↗
PubMed1999

Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation.

The amyloid-beta precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-beta (A beta) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimer's disease brains and colocalization of its APP cleavage product with A beta in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for A beta peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimer's disease.

Acute Disease↗
PubMed1999

Pancreatic islet autotransplantation combined with total pancreatectomy for the treatment of chronic pancreatitis--the Leicester experience.

Islet autotransplantation offers the potential for preventing the surgically induced diabetes that is an inevitable consequence of total pancreatectomy. This paper describes the first islet autotransplant programme in the United Kingdom and the first series in the world to use the spleen as a site for the islet graft. Over an 11 month period, 7 patients underwent total pancreatectomy for chronic pancreatitis combined with a simultaneous islet autotransplant. All 7 patients had normal glucose-tolerance levels and normal C-peptide levels pre-operatively. In 6 patients, islets were embolized into the liver via the portal vein (median transplanted volume=8.5 ml). In addition, 3 patients received islets into the splenic sinusoids via a short gastric vein (median transplanted volume=4 ml). One patient received islets into the spleen alone. One patient died of a stroke 4 weeks post transplantation. Two patients have achieved insulin independence, with a further two patients achieving "transient" insulin independence (<1 month). The remaining 2 patients, although requiring reduced insulin doses, have not achieved insulin-independence. However, all patients have C-peptide levels within the normal range. In trying to explain these findings, split proinsulin levels were measured and found to be elevated. High levels of split proinsulin cross react with the C-peptide assay and this would explain the falsely elevated C-peptide levels. Indeed insulin levels in these patients were all below the normal range. These findings would suggest that the use of C-peptide levels as the "gold standard" for monitoring islet autograft function, may require reappraisal.

C-Peptide↗
PubMed1999

Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons.

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.

Adenoviridae↗
PubMed1998

Immediate-early gene expression in the brain of the thiamine-deficient rat.

Pyrithiamine-induced thiamine deficiency (PTD) in the rat is associated with neuronal loss in the thalamus and inferior colliculus. Recently, we were able to demonstrate the occurrence of apoptosis in the thalamus of these animals. Given that immediate-early genes (IEGs) participate in signal transduction pathways that mediate programmed cell death, the present study utilized in situ hybridization and immunohistochemistry to examine the expression of four IEGs (c-fos, c-jun, fos-B, and NGFI-A) during the progression of PTD. Elevated c-fos mRNA levels were initially observed in the posterior medial thalamus on d 12 of the deficiency. At the acute symptomatic stage (characterized by a loss of righting reflex on d 16-17), the posterior-medial thalamus exhibited increased mRNA for all genes examined, whereas the inferior colliculus demonstrated mRNA induction for c-fos, c-jun, and NGFI-A. Immunohistochemical analysis revealed that elevations of IEG mRNA associated with the acute symptomatic stage were consistently translated into protein in the thalamus. In contrast, whereas elevated Fos- and Jun-like immunoreactivity were detected in the inferior colliculus at this stage, NGFI-A-like immunoreactivity declined significantly below basal levels, suggesting a translational block. These results are consistent with our recent findings of apoptotic cell death, and indicate that differential patterns of IEG expression occur in the thalamus and inferior colliculus during PTD, which may contribute to the pathogenesis of this disorder.

Animals↗
PubMed1998

D1-Receptor-related priming is attenuated by antisense-meditated 'knockdown' of fosB expression.

Administration of dopamine receptor agonists to rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway produce changes in the denervated striatum that enable a subsequent injection to elicit more vigorous circling. The molecular basis for this behavioural phenomenon, termed priming, is unknown. D1-receptor-related priming has been associated with a profound elevation of immediate-early gene (IEG) expression in the denervated striatum. Since immediate-early genes encode known transcriptional regulating factors, this observation has led to the suggestion that IEG induction may play a role in the gene signaling pathways which mediate priming. In the present study, we addressed the role of induction of the IEG fosB in dopamine agonist-induced priming by examining whether inhibition of the synthesis of FosB proteins (FosB and DeltaFosB) by intrastriatal delivery of an antisense oligonucleotide to fosB reduced apomorphine-induced priming. Intrastriatal delivery of an antisense, but not a random, oligonucleotide to fosB 18 and 6 h before apomorphine reduced the ability of this mixed D1¿D2-like receptor agonist to prime circling induced by the specific D1-like receptor agonist SKF 38393. Immunohistochemical analysis revealed that only the antisense oligonucleotide blocked apomorphine-induced increases in FosB-like immunoreactivity in the denervated striatum. In contrast, apomorphine-induced increases in JunB-, NGFI-A- and Fos2-16-like immunoreactivities were unaffected by either the antisense or random oligonucleotides, indicating that the antisense oligonucleotide attenuated apomorphine-induced priming by selectively blocking the synthesis of FosB proteins. Taken together, these findings suggest that fosB induction in the denervated striatum plays a role in mediating D1-receptor-related priming. Dopamine replacement therapy for Parkinson's disease is often complicated by the development of dyskinetic side effects. Results from the present study suggest that D1-receptor-mediated increases in fosB expression may be involved in those intracellular events responsible for the generation of these debilitating side effects.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1998

Hippocampal Myc and p53 expression following transient global ischemia.

The proto-oncogene c-myc, and the tumor suppressor gene p53, encode proteins which function as transcriptional regulating factors governing cell proliferation, differentiation, and apoptosis. Recent evidence suggests that the delayed neuronal death which follows an episode of transient forebrain ischemia may involve apoptotic processes. We have therefore utilized immunohistochemistry to investigate the effects of transient global ischemia on neuronal expression of p53- and Myc-like immunoreactivities in the rodent forebrain 2, 12, 24, 48, and 72 h following reperfusion. Transient global ischemia (20 min), produced by four vessel occlusion (4-VO), initially elevated p53-like immunoreactivity in both CA1 and CA3 hippocampal subfields at 24 h of recirculation. However, distinct patterns of gene expression became evident in these regions at later time points. A pivotal difference was the persistence of ischemia-induced increases of p53- and Myc-like immunoreactivity in the CA1 region of the hippocampus. Unlike CA3 neurons where p53-like immunoreactivity subsided to basal levels by 48 h of survival, CA1 neurons continued to display increased p53-immunoreactivity 48 h post-ischemia, while Myc-like immunoreactivity was selectively elevated in CA1 neurons at this time point. Ischemia-induced increases in p53-like immunoreactivity were also detected in vulnerable regions of the amygdala, thalamus, and cortex 12 to 48 h after recirculation. Given that both p53 and Myc have been implicated in gene signalling pathways which mediate programmed cell death, our findings which demonstrate that 4-VO produces persistent elevations of p53- and Myc-like immunoreactivities in vulnerable neurons suggest that these proteins may also contribute to delayed neuronal death following an episode of transient forebrain ischemia.

Amygdala↗
PubMed1998

Ischemia-induced CA1 neuronal death is preceded by elevated FosB and Jun expression and reduced NGFI-A and JunB levels.

Alterations in levels of the immediate-early gene (IEG) proteins Fos, FosB, DeltaFosB, Jun, JunB, JunD, and NGFI-A were investigated in rat hippocampus by immunohistochemistry 2, 12, 24, and 48 h after forebrain ischemia. Transient global ischemia of 20 min, produced by four vessel occlusion (4-VO), elicited different patterns of IEG expression in vulnerable CA1 and more resilient CA3 neurons. Cell counts revealed that except for JunD and NGFI-A, immunoreactivity for all examined IEGs was initially elevated by forebrain ischemia in both CA1 and CA3 hippocampal subfields. However, distinct patterns of IEG expression became evident in these regions at later time points. The pivotal difference was the persistence of ischemia-induced elevations of FosB and Jun expression in the CA1 region of the hippocampus. Unlike CA3 neurons, where IEG immunoreactivity had subsided to basal levels by 24-48 h, CA1 neurons continued to display increased FosB- and Jun-like immunoreactivity 48 h post-ischemia. Western blot analysis revealed that elevated expression of both FosB and DeltaFosB-like proteins were responsible for the immunohistochemical detection of enhanced FosB-like immunoreactivity in CA1 neurons at 48 h. These findings are consistent with recent in vitro studies that implicate FosB and Jun in gene signalling pathways responsible for programmed cell death. In contrast to FosB and Jun, JunB expression declined significantly below basal levels in CA1 neurons at 48 h, yet remained unaltered in CA3 neurons. Given that JunB can inhibit the transactivating properties of Jun, decreased JunB levels may contribute to the apoptotic death of CA1 neurons by enhancing the transcriptional regulating activity of Jun. Also notable at 48 h was the complete loss of constitutive NGFI-A expression from CA1 neurons of ischemic animals. These findings suggest that persistent elevations in FosB and Jun expression, concurrent with reductions in JunB and NGFI-A levels, contribute to the apoptotic death of CA1 neurons after forebrain ischemia.

Animals↗
PubMed1998

Experimental study of electrolysis-induced hepatic necrosis.

BACKGROUND: One of the most promising but unexplored methods for treating patients with irresectable liver tumours is electrolysis. This study examined the effect of increasing 'current dose' on the volume of the lesion induced in normal rat liver. METHODS: A direct current generator, connected to platinum electrodes implanted in the rat liver, was used to examine the effect of (1) varying current doses from 1 to 5 coulombs and (2) electrode separation (2 or 20 mm), on the volume of liver necrosis. RESULTS: There was a significant correlation (P < 0.001) between the current dose and the volume of necrosis produced for each electrode separation. Placing the electrodes 2 mm apart resulted in smaller total volumes of necrosis than placing them 20 mm apart when anode lesions were significantly larger than cathode lesions (P< 0.05). Liver enzymes (aspartate aminotransferase, alanine aminotransferase) were significantly raised 1 day after treatment (P < 0.001) and predicted the total volume of hepatic necrosis (P < 0.001). CONCLUSION: Predictable and reproducible areas of liver necrosis are produced with electrolysis. If these results extrapolate to larger animal models, this technique has potential for patients with irresectable primary and secondary liver tumours.

Animals↗
PubMed1998

Increased Mdm2 expression in rat brain after transient middle cerebral artery occlusion.

The negative regulator of p53 transactivation, Mdm2, increased in the ischemic territory after 90 minutes of transient middle cerebral artery occlusion in spontaneously hypertensive rats compared to sham controls. Increased mdm2 mRNA was detected by semiquantitative reverse transcriptase polymerase chain reaction by 6 hours of reperfusion in the ipsilateral hemisphere. In situ hybridization histochemistry was used to localize increases in mdm2 mRNA which occurred in neurons of ischemic cortex and dorsolateral striatum. The number of labeled neurons increased by approximately 20-fold and the cells displayed five-fold increases of mdm2 mRNA in the cortex. Immunohistochemical staining for Mdm2 revealed that its mRNA was efficiently translated in the ischemic cortex, but not striatum, by 8 to 24 hours of reperfusion. Western blotting confirmed 30- to 40-fold increases in the full-length protein of 90 kd at these time points without evidence of alternative splicing. Because Mdm2 is a negative regulator of the apoptosis promoting activity of p53, increased expression of Mdm2 may be a component of a repair response in injured neurons, and supports Mdm2 being an indicator of DNA damage in the brain early after an ischemic insult in a similar way to Gadd45.

Animals↗
PubMed1998

The best management for 'crescendo biliary colic' is urgent laparoscopic cholecystectomy.

Gallbladder disease due to stones is well recognised as falling into two categories, presenting with either chronic symptoms or developing acute cholecystitis or other complications. We describe an intermediate group of 14 patients (11 women, three men, median age 31 years) presenting with 4-14 days of at least daily attacks of resolving biliary colic, who underwent early laparoscopic cholecystectomy within 24 hours of presentation. None had any evidence of acute inflammation, either at laparoscopy or on histology. Their surgery was straightforward with operating times ranging from 35-80 minutes and no complications. Patients with 'crescendo biliary colic' are often young women who can rarely afford invalidity. Rather than the current practice of analgesia for each attack and elective surgery weeks later, they are optimally managed by urgent laparoscopic cholecystectomy, preventing the development of complications and minimising the need for further medical involvement.

Adult↗
PubMed1997

Distribution of neuronal apoptosis inhibitory protein-like immunoreactivity in the rat central nervous system.

We have recently shown that spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by motor neuron loss, is associated with deletion of a gene that encodes the neuronal apoptosis inhibitory protein (NAIP). In the present study, we have examined the distribution of NAIP-like immunoreactivity (NAIP-LI) in the rat central nervous system (CNS) by using an affinity-purified polyclonal antibody against NAIP. In the forebrain, immunoreactive neurons were detected in the cortex, the hippocampus (pyramidal cells, dentate granule cells, and interneurons), the striatum (cholinergic interneurons), the basal forebrain (ventral pallidum, medial septal nucleus, and diagonal band), the thalamus (lateral and ventral nuclei), the habenula, the globus pallidus, and the entopenduncular nucleus. In the midbrain, NAIP-LI was located primarily within neurons of the red nucleus, the substantia nigra pars compacta, the oculomotor nucleus, and the trochlear nucleus. In the brainstem, neurons containing NAIP-LI were observed in cranial nerve nuclei (trigeminal, facial, vestibular, cochlear, vagus, and hypoglossal nerves) and in relay nuclei (pontine, olivary, lateral reticular, cuneate, gracile nucleus, and locus coeruleus). In the cerebellum, NAIP-LI was found within both Purkinje and nuclear cells (interposed and lateral nuclei). Finally, within the spinal cord, NAIP-LI was detected in Clarke's column and in motor neurons. Taken together, these results indicate that NAIP-LI is distributed broadly in the CNS. However, high levels of NAIP-LI were restricted to those neuronal populations that have been reported to degenerate in SMA. This anatomical correspondence provides additional evidence for NAIP involvement in the neurodegeneration observed in acute SMA.

Animals↗
PubMed1997

Local transcriptional control of utrophin expression at the neuromuscular synapse.

Recently, the use of a transgenic mouse model system for Duchenne muscular dystrophy has demonstrated the ability of utrophin to functionally replace dystrophin and alleviate the muscle pathology (see Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349-353). However, there is currently a clear lack of information concerning the regulatory mechanisms presiding over utrophin expression during normal myogenesis and synaptogenesis. Using in situ hybridization, we show that utrophin mRNAs selectively accumulate within the postsynaptic sarcoplasm of adult muscle fibers. In addition, we demonstrate that a 1.3-kilobase fragment of the human utrophin promoter is sufficient to confer synapse-specific expression to a reporter gene. Deletion of 800 base pairs from this promoter fragment reduces the overall expression of the reporter gene and abolishes its synapse-specific expression. Finally, we also show that utrophin is present at the postsynaptic membrane of ectopic synapses induced to form at sites distant from the original neuromuscular junctions. Taken together, these results indicate that nerve-derived factors regulate locally the transcriptional activation of the utrophin gene in skeletal muscle fibers and that myonuclei located in extrasynaptic regions are capable of expressing utrophin upon receiving appropriate neuronal cues.

Animals↗