Ethanol feeding induces insulin resistance with enhanced PI 3-kinase activation

High ethanol intake is considered to impair insulin sensitivity. In the present study, we investigated the acute and chronic effects of ethanol intake on glucose metabolism and insulin signal transduction. Hyperinsulinemic-euglycemic clamp studies revealed 70% and 51% decreases in the glucose infusion rate, 52% and 31% decreases in the glucose utilization rate, and 6.6- and 8.0-fold increases in hepatic glucose in continuous- and acute-ethanol-loaded rats, respectively. Despite the presence of insulin resistance, alcohol-fed rats showed enhanced tyrosine phosphorylation of insulin receptors, IRS-1 and IRS-2, induced by insulin injection via the portal vein. PI 3-kinase activities associated with IRSs and phosphotyrosine also increased significantly as compared with those of controls. These data suggest ethanol intake to be a factor leading to insulin resistance, regardless of whether it is a single or continuous intake. In addition, the insulin signaling step impaired by ethanol feeding is likely to be downstream from PI 3-kinase.     

High expression of Cks1 in human non-small cell lung carcinomas

Enhanced degradation of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is known to be a powerful prognostic marker in many types of human cancers. Human CDK subunit 1 (Cks1) and S-phase kinase associated protein 2 (Skp2) are components of the SCF(Skp2) complex, which acts as a ubiquitin ligase for p27(Kip1). There are no reports about the involvement of Cks1 in the pathogenesis of human cancer. Here we show high expression of Cks1 in non-small cell lung cancers (NSCLCs) using Western blotting and quantitative real-time RT-PCR. The Skp2 mRNA expression level was high in squamous cell carcinomas and was inversely related with the p27(Kip1) protein level in individual clinical samples. In contrast, Cks1 mRNA expression had no such relationship with p27(Kip1), although Cks1 mRNA was significantly elevated in adenocarcinomas. These results suggest that high expression of Skp2 and Cks1 may be involved in the pathogenesis of NSCLCs via different mechanisms.     

Facilitated angiogenesis induced by heme oxygenase-1 gene transfer in a rat model of hindlimb ischemia

Heme oxygenase-1 (HO-1) is an inducible form of heme oxygenase that catabolizes heme to carbon monoxide, biliverdin, and ferrous iron. We have investigated whether HO-1 can induce angiogenic effects in vivo. Rats were subjected to a bolus injection of either wild type adenovirus (ad-wt) or adenovirus encoding HO-1 (ad-HO-1) through the right femoral artery, which was then removed immediately. HO-1 gene transfer resulted in about a sixfold increase in HO-1 protein levels as compared to the non-treated animals. The increase in both blood flow and capillary density was significantly greater in the ischemic hindlimbs that had been injected with ad-HO-1 than in those injected with ad-wt. These angiogenic effects of ad-HO-1 infection could be completely abolished by treating the animals with the HO inhibitor, zinc protoporphyrin, indicating that they were specifically due to the expression of HO-1. Thus, HO-1 gene transfer improves the blood flow in ischemic hindlimb, at least in part, via angiogenesis facilitated by the induction of this molecule.     

Nicotinamide Attenuates Focal Ischemic Brain Injury in Rats: With Special Reference to Changes in Nicotinamide and NAD+ Levels in Ischemic Core and Penumbra

We investigated the neuroprotective action of nicotinamide in focal ischemia. Male spontaneously hypertensive rats (5–7 months old) were subjected to photothrombotic occlusion of the right distal middle cerebral artery (MCA). Either nicotinamide (125 or 250 mg/kg) or vehicle was injected IV before MCA occlusion. Changes in the cerebral blood flow (CBF) were monitored using laser-Doppler flowmetry, and infarct volumes were determined with TTC staining 3 days after MCA occlusion. In another set of experiments, the brain nicotinamide and nicotinamide adenine dinucleotide (NAD⁺) levels were analyzed by HPLC using the frozen samples dissected from the regions corresponding to the ischemic core and penumbra. In the 250-mg/kg nicotinamide group, the ischemic CBF was significantly increased compared to that the untreated group, and the infarct volumes were substantially attenuated (–36%). On the other hand, the ischemic CBF in the 125 mg/kg nicotinamide group was not significantly different from the untreated CBF, however, the infarct volumes were substantially attenuated (–38%). Cerebral ischemia per se did not affect the concentrations of nicotinamide and NAD⁺ both in the penumbra and ischemic core. In the nicotinamide groups, the brain nicotinamide levels increased significantly in all areas examined, and brain NAD⁺ levels increased in the penumbra but not in the ischemic core. Increased brain levels of nicotinamide are considered to be primarily important for neuroprotection against ischemia, and the protective action may be partly mediated through the increased NAD⁺ in the penumbra.     

Identification of a beta-secretase activity,which truncates amyloid beta-peptide after its presenilin-dependent generation

The beta-amyloid precursor protein (beta APP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid beta-peptide (A beta). N-terminal cleavage is mediated by beta-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) gamma-secretase complex. The A beta-generating gamma-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of A beta-(1-40) or A beta-(1-42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted A beta-(1-40/42). However, mass spectrometry revealed a truncated A beta species, which terminates at amino acid 34 (A beta-(1-34)) suggesting an alternative gamma-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of A beta-(1-40) and A beta-(1-42) but also that of A beta-(1-34). However, expression levels of BACE correlate with the amount of A beta-(1-34), and A beta-(1-34) is produced at the expense of A beta-(1-40) and A beta-(1-42). Since this suggested that BACE is involved in a C-terminal truncation of A beta, we incubated purified BACE with A beta-(1-40) in vitro. Under these conditions A beta-(1-34) was generated. Moreover, when conditioned media containing Abeta-(1-40) and A beta-(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, A beta-(1-34) was efficiently produced in vivo. These data demonstrate that an apparently gamma-secretase-dependent A beta derivative is produced after the generation of the non-truncated A beta via an additional and unexpected activity of BACE.     

The Wilhelmine E. Key 2001 Invitational Lecture. Estimation of divergence times for a few mammalian and several primate species

Statistical methods for estimating divergence times by using multiprotein gamma distances are discussed. When a large number of proteins are used, even a small degree of deviation from the molecular clock hypothesis can be detected. In this case, one may use the stem-lineage method for estimating divergence times. However, the estimates obtained by this method are often similar to those obtained by the linearized tree method. Application of these methods to a dataset of 104 proteins from several vertebrate species indicated that the divergence times between humans and mice and between mice and rats are about 96 and 33 million years (MY) ago, respectively. These estimates were obtained by assuming that birds and mammals diverged 310 MY ago. Similarly application of the methods to the protein sequence data from primate species indicated that the human lineage separated from the chimpanzee, gorilla, Old World monkeys, and New World monkeys about 6.0, 7.0, 23.0, and 33.0 MY ago, respectively. In this case the use of two calibration points, that is, the divergence time (13 MY ago) between humans and orangutans and between primates and artiodactyls (90 MY ago) gave essentially the same estimates.     

The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis

Matrix metalloproteinases (MMPs) are a large group of enzymes responsible for matrix degradation. Among them, the family of gelatinases (MMP-2/gelatinase A and MMP-9/gelatinase B) is overproduced in the joints of patients with rheumatoid arthritis. Because of their degradative effects on the extracellular matrix, gelatinases have been believed to play an important role in progression and cartilage degradation in this disease, although their precise roles are yet to be defined. To clarify these roles, we investigated the development of Ab-induced arthritis, one of the murine models of rheumatoid arthritis, in MMP-2 or MMP-9 knockout (KO) mice. Surprisingly, the MMP-2 KO mice exhibited severe clinical and histologic arthritis than wild-type mice. The MMP-9 KO mice displayed milder arthritis. Recovery from exacerbated arthritis in the MMP-2 KO mice was possible by injection of wild-type fibroblasts. These results indicated a suppressive role of MMP-2 and a pivotal role of MMP-9 in the development of inflammatory joint disease.     

Comparison of arbitrarily primed-polymerase chain reaction and pulse-field gel electrophoresis for characterizing methicillin-resistant Staphylococcus aureus

AIMS: The aim of this study was to analyse genotypes for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including hetero-vancomycin-resistant Staph. aureus (VRSA), at a Japanese university hospital. METHODS AND RESULTS: Seventy-eight clinical isolates of MRSA were analysed by arbitrarily primed-polymerase chain reaction (AP-PCR) using ERIC2 primer and by pulse-field gel electrophoresis (PFGE) following SmaI digestion. Analyses of the nine genotypes and 28 subtypes defined by PFGE, and of the three genotypes and 22 subtypes defined by AP-PCR, both facilitated epidemiological tracing. Used in combination, AP-PCR and PFGE provided more precise classification than the use of a single genotyping method. The six hetero-VRSA isolates were classified into four genotypes defined by the combination of both methods, but these genotypes contained non-VRSA isolates. CONCLUSIONS: The results suggest that both PFGE and AP-PCR are useful in discriminating MRSA, but not hetero-VRSA, isolates for epidemiological analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining the results of PFGE with the results of AP-PCR can provide more detail differentiation of MRSA and hetero-MRSA isolates than either method alone.     

Purifying selection and birth-and-death evolution in the histone H4 gene family

Histones are small basic proteins encoded by a multigene family and are responsible for the nucleosomal organization of chromatin in eukaryotes. Because of the high degree of protein sequence conservation, it is generally believed that histone genes are subject to concerted evolution. However, purifying selection can also generate a high degree of sequence homogeneity. In this study, we examined the long-term evolution of histone H4 genes to determine whether concerted evolution or purifying selection was the major factor for maintaining sequence homogeneity. We analyzed the proportion (p(S)) of synonymous nucleotide differences between the H4 genes from 59 species of fungi, plants, animals, and protists and found that p(S) is generally very high and often close to the saturation level (p(S) ranging from 0.3 to 0.6) even though protein sequences are virtually identical for all H4 genes. A small proportion of genes showed a low level of p(S) values, but this appeared to be caused by recent gene duplication. Our findings suggest that the members of this gene family evolve according to the birth-and-death model of evolution under strong purifying selection. Using histone-like genes in archaebacteria as outgroups, we also showed that H1, H2A, H2B, H3, and H4 histone genes in eukaryotes form separate clusters and that these classes of genes diverged nearly at the same time, before the eukaryotic kingdoms diverged.