Antitumor activity of a small-molecule inhibitor of the histone kinase Haspin.

The approval of histone deacetylase inhibitors for treatment of lymphoma subtypes has positioned histone modifications as potential targets for the development of new classes of anticancer drugs. Histones also undergo phosphorylation events, and Haspin is a protein kinase the only known target of which is phosphorylation of histone H3 at Thr3 residue (H3T3ph), which is necessary for mitosis progression. Mitotic kinases can be blocked by small drugs and several clinical trials are underway with these agents. As occurs with Aurora kinase inhibitors, Haspin might be an optimal candidate for the pharmacological development of these compounds. A high-throughput screening for Haspin inhibitors identified the CHR-6494 compound as being one promising such agent. We demonstrate that CHR-6494 reduces H3T3ph levels in a dose-dependent manner and causes a mitotic catastrophe characterized by metaphase misalignment, spindle abnormalities and centrosome amplification. From the cellular standpoint, the identified small-molecule Haspin inhibitor causes arrest in G2/M and subsequently apoptosis. Importantly, ex vivo assays also demonstrate its anti-angiogenetic features; in vivo, it shows antitumor potential in xenografted nude mice without any observed toxicity. Thus, CHR-6494 is a first-in-class Haspin inhibitor with a wide spectrum of anticancer effects that merits further preclinical research as a new member of the family of mitotic kinase inhibitors.

Identification of potential stroke targets by lentiviral vector mediated overexpression of HIF-1 alpha and HIF-2 alpha in a primary neuronal model of hypoxia.

The identification of genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. Furthermore, the characterization of such pathways could facilitate the identification of novel targets for stroke therapy. We have used a novel approach to amplify differential gene expression patterns in a primary neuronal model of stroke by employing a lentiviral vector system to specifically bias the transcriptional activation of hypoxically regulated genes. Overexpression of the hypoxia-induced transcription factor subunits HIF-1 alpha and HIF-2 alpha elevated hypoxia-mediated transcription of many known HIF-regulated genes well above control levels. Furthermore, many potentially novel HIF-regulated genes were discovered that were not previously identified as hypoxically regulated. Most of the novel genes identified were activated by a combination of HIF-2 alpha overexpression and hypoxic insult. These included several genes with particular importance in cell survival pathways and of potential therapeutic value. Hypoxic induction of HIF-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. Further investigation of the genes identified in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia.

Sequence variation in the early genes E1E4, E6 and E7 of human papilloma virus type 6.

The majority of condylomata acuminata (anogenital warts) are caused by infection with Human Papilloma Virus type 6 (HPV-6). We have sequenced the HPV-6 early genes, E1-E4, E6 and E7 from wart biopsy DNA samples sourced from the UK and USA and derived a consensus sequence for these genes and the proteins they encode. When compared to the prototype HPV-6b sequence, published over 12 years ago, the E1-E4 consensus sequence showed 3/91 (3.3%) amino acid changes, the E6 consensus sequence showed 1/150 (0.7%) changes and the E7 consensus sequence showed 1/98 (1.0%) changes. Since many of the early gene sequences from biopsy material were more similar to the HPV-6a subtype than HPV-6b, this data supports the use of HPV-6a as the HPV-6 prototype.

A 31P magnetic resonance spectroscopy study of mitochondrial function in skeletal muscle of patients with Parkinson's disease.

The activity of complex I of the respiratory chain is decreased in the substantia nigra of patients with Parkinson's disease (PD) but the presence of this defect in skeletal muscle is controversial. Therefore, the mitochondrial function of skeletal muscle in patients with PD was investigated in vivo using 31P magnetic resonance spectroscopy. Results from 7 PD patients, 11 age matched controls and 9 mitochondrial myopathy patients with proven complex I deficiency were obtained from finger flexor muscle at rest, during exercise and in recovery from exercise. In resting muscle, the patients with mitochondrial myopathy showed a low PCr/ATP ratio, a low phosphorylation potential, a high P(i)/PCr ratio and a high calculated free [ADP]. During exercise, stores of high energy phosphate were depleted more rapidly than normal, while in recovery, the concentration of phosphocreatine and free ADP returned to pre-exercise values more slowly than normal. In contrast, the patients with PD were not significantly different from normal for any of these variables, and no abnormality of muscle energetics was detected. Three of the PD patients also had mitochondrial function assessed biochemically in muscle biopsies. No respiratory chain defect was identified in any of these patients by polarography or enzyme analysis when compared with age-matched controls. These results suggest that skeletal muscle is not a suitable tissue for the investigation and identification of the biochemical basis of the nigral complex I deficiency in PD.

Platelet mitochondrial function in Parkinson's disease. The Royal Kings and Queens Parkinson Disease Research Group.

There is increasing evidence that defective function of the mitochondrial enzyme NADH CoQ reductase (complex I) is involved not only in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity, but also in idiopathic Parkinson's disease (PD). Complex I deficiency has been identified in PD substantia nigra and appears to be disease-specific and selective for the substantia nigra within the central nervous system. We describe a method for preparation of an enriched mitochondrial fraction from 60 mL blood. Using this technique, we analyzed respiratory chain function in 25 patients with PD and 15 matched control subjects. We confirm a previous report of a specific complex I deficiency in PD platelet mitochondria. Although there was a statistically significant decrease in complex I activity in the PD group compared with the control group (p = 0.005), the defect was mild (16%); it was not possible to distinguish PD from control values on an individual basis. This deficiency is not detectable in platelet whole-cell homogenates, presumably reflecting the relative insensitivity of this preparation and the limited decrease in complex I activity in PD. The presence of a mild complex I defect in platelets together with a more severe defect in substantia nigra suggests either that the pharmacological characteristics shared by these two tissues render them susceptible to a particular toxin or toxins, or that the defect is widely distributed and other biochemical events enhance the deficiency in substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)

Human mitochondrial complex I dysfunction.

In humans, complex I dysfunction has been observed in a high percentage of patients with mitochondrial myopathy. Analysis of mitochondria from these patients suggests the function and assembly of complex I is particularly susceptible to abnormalities of mitochondrial DNA, involving either point mutations of tRNA genes or major deletions. The evidence for a complex I defect in Parkinson's disease is accumulating, although the cause of this deficiency or the role it plays in the events that culminate in dopaminergic cell death remains unresolved.

Brain, skeletal muscle and platelet homogenate mitochondrial function in Parkinson's disease.

The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with idiopathic Parkinson's disease has provided new understanding into the possible mechanisms that may underlie this neurodegenerative disorder. The biochemical defect is identical to that induced in humans, primates and mice exposed to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We have studied mitochondrial respiratory chain function in various brain regions, in skeletal muscle and in blood platelets from patients with idiopathic Parkinson's disease and from matched controls. We provide evidence suggesting that the complex I deficiency in Parkinson's disease is limited to the brain and that this defect is specific for the substantia nigra. The tissue specificity of the complex I deficiency in Parkinson's disease and its localization to the substantia nigra support the proposition that complex I deficiency may be directly involved in the cause of dopaminergic cell death in Parkinson's disease. An understanding of the molecular basis of this biochemical defect will provide valuable insight into the cause of Parkinson's disease. Our findings of normal mitochondrial function in platelet homogenates suggests that this tissue cannot be used to develop a 'diagnostic test' for Parkinson's disease.

Mitochondrial function in Parkinson's disease. The Royal Kings and Queens Parkinson's Disease Research Group.

There is increasing evidence for a defect of mitochondrial respiratory chain function in Parkinson's disease. Specific NADH CoQ1 reductase (complex I) deficiency has been identified in the substantia nigra. Available evidence suggests that this defect is confined to the substantia nigra and is not present elsewhere in the parkinsonian brain. The absence of a detectable mitochondrial abnormality in the substantia nigra of patients with multiple system atrophy also suggests that the complex I deficiency in Parkinson's disease is not simply due to an artifact of neuronal degeneration. Evidence for abnormal mitochondrial function in skeletal muscle is conflicting; two studies showed multiple respiratory chain defects and one study was unable to demonstrate any deficiency. A severe deficiency of complex I activity has been found in platelet mitochondria from parkinsonian patients. This finding has not as yet been confirmed. Platelet homogenates do not show the complex I deficiency, however, suggesting that such a preparation may be too insensitive to detect the defect. The role of complex I deficiency in the events that culminate in dopaminergic cell death in Parkinson's disease remains unresolved. It is likely that if this mitochondrial defect is confirmed, it will be related to a number of other factors, including environmental agents, oxidative stress, and genetic predisposition.

The use of a geographical information system for hospital catchment area research in Natal/KwaZulu.

We use a computerised geographical information system (GIS) to study the population per bed ratios and the implications of open access to the private and the formerly white hospital services in Natal. The advantages of the GIS method over the more usual administrative boundary-based beds per capita ratios are discussed. While the latter method would suggest that hospital bed resources in the province are racially unequal but nevertheless adequate (264 people per general and referral bed for the whole population, 195 for whites and 275 for blacks) the GIS analysis reveals widespread inadequacy, worse for blacks. Of the estimated hospital catchment areas half have more than 275 black people per general and referral bed, and half of these have more than 550 black people per bed. One-third of the catchment areas estimated for whites have ratios above 275 people per bed, and one half of these are also above 550 people per bed. The GIS analysis shows that open access to beds previously reserved for whites will make no difference to rural blacks, and almost none to urban blacks, because there were relatively few such beds, and they were concentrated in the cities. For the same reasons, the opening of private hospital beds to all patients would not significantly alleviate the apparent bed shortages in priority areas. By contrast, people in these priority areas would gain significantly improved access to general hospital care if selected chronic disease and industrial hospitals were upgraded to provide general hospital services.(ABSTRACT TRUNCATED AT 250 WORDS)