Renal arteriolar hyalinosis, not intimal thickening in large arteries, is associated with cardiovascular events in people with biopsy-proven diabetic nephropathy.

AIMS: Diabetic nephropathy, a pathologically diagnosed microvascular complication of diabetes, is a strong risk factor for cardiovascular events, which mainly involve arteries larger than those affected in diabetic nephropathy. However, the association between diabetic nephropathy pathological findings and cardiovascular events has not been well studied. We aimed to investigate whether the pathological findings in diabetic nephropathy are closely associated with cardiovascular event development. METHODS: This retrospective cohort study analysed 377 people with type 2 diabetes and biopsy-proven diabetic nephropathy, with a median follow-up of 5.9 years (interquartile range 2.0 to 13.5). We investigated how cardiovascular events were impacted by two vascular diabetic nephropathy lesions, namely arteriolar hyalinosis and arterial intimal thickening, and by glomerular and interstitial lesions. RESULTS: Of the 377 people with diabetic nephropathy, 331 (88%) and 295 (78%) had arteriolar hyalinosis and arterial intimal thickening, respectively. During the entire follow-up period, those with arteriolar hyalinosis had higher cardiovascular event rates in the crude Kaplan-Meier analysis than those without these lesions (P = 0.005, log-rank test). When fully adjusted for clinically relevant confounders, arteriolar hyalinosis independently predicted cardiovascular events [hazard ratio (HR) 1.99; 95% confidence interval (CI) 1.12, 3.86], but we did not find any relationship between arterial intimal thickening and cardiovascular events (HR 0.89; 95% CI 0.60, 1.37). Additionally, neither glomerular nor interstitial lesions were independently associated with cardiovascular events in the fully adjusted model. CONCLUSIONS: Arteriolar hyalinosis, but not intimal thickening of large arteries, was strongly associated with cardiovascular events in people with diabetic nephropathy.

Developmental alterations in expression and subcellular localization of antizyme and antizyme inhibitor and their functional importance in the murine mammary gland.

Ornithine decarboxylase (ODC), antizyme (AZ), and antizyme inhibitor (AIn) play a key role in regulation of intracellular polyamine levels by forming a regulatory circuit through their interactions. To gain insight into their functional importance in cell growth and differentiation, we systematically examined the changes of their expression, cellular polyamine contents, expression of genes related to polyamine metabolism, and beta-casein gene expression during murine mammary gland development. The activity of ODC and AZ1 as well as putrescine level were low in the virgin and involuting stages, but they increased markedly during late pregnancy and early lactation when mammary cells proliferate extensively and begin to augment their differentiated function. The level of spermidine and expression of genes encoding spermidine synthase and AIn increased in a closely parallel manner with that of casein gene expression during pregnancy and lactation. On the other hand, the level of spermidine/spermine N(1)-acetyltransferase (SSAT) mRNA and AZ2 mRNA decreased during those periods. Immunohistochemical analysis showed the translocation of ODC and AIn between the nucleus and cytoplasm and the continuous presence of AZ in the nucleus during gland development. Reduction of AIn by RNA interference inhibited expression of beta-casein gene stimulated by lactogenic hormones in HC11 cells. In contrast, reduction of AZ by AZsiRNA resulted in the small increase of beta-casein gene expression. These results suggested that AIn plays an important role in the mammary gland development by changing its expression, subcellular localization, and functional interplay with AZ.

Antioxidant and antimicrobial effects of garlic in chicken sausage.

The antioxidant and antimicrobial effects of equivalent concentrations of fresh garlic (FG), garlic powder (GP) and garlic oil (GO) were investigated against lipid oxidation and microbial growth in raw chicken sausage during storage at 3 degrees C. The antioxidant activities were compared to that of a standard synthetic antioxidant; butylated hydroxyanisole (BHA). The initial mean levels of thiobarbituric acid (TBA) value and peroxide value (POV) were 0.140 and 6.32, respectively. However after 21 days of storage, TBA and POV ranged from 0.151 to 4.92, respectively, in FG (50 g/kg) formulated samples to 0.214 and 8.64, respectively, in GO (0.06 g/ kg) formulation. Addition of either garlic or BHA (0.1 g/kg) significantly delayed lipid oxidation when compared with control. The antioxidant activities of the various materials added followed the order FG>GP>BHA>GO. On the other hand, the initial aerobic plate count (APC) in the samples was 4.41 log(10) CFU/g. Addition of FG (30 g/kg) or GP (9 g/kg) significantly reduced the APC and, subsequently, the shelf-life of the product was extended to 21 days. However, addition of GO or BHA resulted in no significant difference in APC when compared with control. Sensory analysis indicated that FG had a significant stronger flavor than the other sausage formulations. The results suggest that fresh garlic and garlic powder, through their combined antioxidant and antimicrobial effects, are potentially useful in preserving meat products.

Microbiological and chemical quality of ground beef treated with sodium lactate and sodium chloride during refrigerated storage.

The effects of sodium lactate (NaL) and sodium chloride (NaCl), either alone (30 g/kg) or in combination (20+20 g/kg), on the microbiological and chemical quality of raw ground beef during vacuum-packaged storage at 2 degrees C were investigated. The results showed that addition of NaL alone or in combination with NaCl significantly delayed the proliferation of aerobic plate counts, psychrotrophic counts, lactic acid bacteria and Enterobacteriaceae and extended the shelf life of the product up to 15 and 21 days, respectively, versus 8 days only for control. Over the storage time (21 days), NaL maintained the ground beef at almost constant pH, while the pH of control or NaCl-treated samples significantly decreased. Lipid oxidation (TBA value) was not affected by addition of NaL. At storage day 21 however, TBA values of both NaL-treated (0.309) and control (0.318) samples were significantly lower than those of samples treated with NaCl (0.463). The combination of NaCl with NaL significantly reduced the oxidative changes caused by NaCl (0.384 versus 0.463). Therefore, NaL alone or in combination with NaCl could be utilized successfully to reduce the microbial growth, maintain the chemical quality, and extend the shelf life of ground beef during refrigerated storage.

CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis.

DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic chromatin condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of chromatin condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic chromatin condensation.

Improved data processing for optical imaging of developing neuronal connectivity in the neonatal mouse barrel cortex.

Optical recording methods using voltage-sensitive dyes have proven valuable for the analysis of neuronal networks both in vivo and in vitro. This technique detects membrane potential changes as changes in the absorption or fluorescence of voltage-sensitive dyes incorporated into the cellular plasma membranes. The reliability of the optical recording technique is dependent on the dye-related response being fast enough to follow the electrical activity and of the response being more or less proportional to the amplitude of the membrane potential change. A high spatial resolution can be achieved using an appropriate imaging system and a dye with a response of sufficiently high signal-to-noise ratio. Thus, it is now anticipated that this method will be able to shed more light on the spatio-temporal information processing of neocortical circuitry. While the FUJIX HR Deltaron 1700 optical imaging system offers a reasonably high time (0.6 ms) and space-resolution (7 microm at 10x magnification), one drawback of this system, however, is its relatively poor data processing capabilities. We have therefore developed a protocol to improve the signal-to-noise ratio by modifying the calculation algorithm of the optical data. Consequently, we characterized optical responses in thalamocortical slices to find developmental landmarks of thalamocortical and intracortical connectivity in the neonatal mouse barrel cortex. Successful application of this method has been published on the analysis of thalamocortical glutamatergic connectivity [8].

Polyamines of the thermophilic eubacteria belonging to the genera Thermosipho, Thermaerobacter and Caldicellulosiruptor.

Cellular polyamines of four new thermophiles located in three early branched eubacterial clades, were investigated for the chemotaxonomic significance of polyamine distribution profiles. The thermophilic anaerobic Thermosipho japonicus, belonging to the order Thermotogales, contained norspermidine, norspermine and thermospermine in addition to spermidine and spermine. The polyamine profile was identical to the polyamine composition of Thermotoga, Fervidobacterium and Petrotoga species of the order. Spermidine, norspermidine, spermine, N4-bis(aminopropyl)spermidine and agmatine were found in thermophilic aerobic Thermaerobacter marianensis. Some differences were observed in the polyamine compositions of the phylogenetically related thermophilic anaerobes, Moorella, Dictyoglomus, Thermoanaerobacterium and Thermoanaerobacter species. Thermophilic anaerobic Caldicellulosiruptor kristianssonii and Caldicellulosiruptor owensensis contained a linear penta-amine, thermopentamine, and two quaternary branched penta-amines, N4-bis(aminopropyl)spermidine and N4-bis(aminopropyl)norspermidine, as the major polyamines. A novel tertiary branched penta-amine, N4-aminopropylspermine, was found in the two Caldicellulosiruptor species.

Measurements of macromolecule-bound and ultra-filtrable polyamines in rat liver homogenized without buffer.

Ultra-filtrable and macromolecule-bound polyamines in rat liver homogenates, made without buffer, were determined, using Potter-Elvehjem homogenizer and commercially available, pressure-aided ultrafiltration device with a membrane pore size that allows passage of particles of molecular weight no larger than 5000. About 90% of polyamines in the liver were shown to be equilibrated with externally added 15N-labeled polyamines, based on the difference in the ratio of the natural to 15N-labeled polyamine in the liver homogenate and the ultrafiltrate. The entire amount of ultrafiltrate in the homogenized liver, required for calculation of the amounts of ultra-filtrable and macromolecule-bound polyamines, was estimated to be about 0.25 g in one gram of the homogenate, using a limited dilution curve of spermine in the ultrafiltrate with phosphate buffered saline and distilled water. With this value, ultra-filtrable polyamines in normal rat liver homogenate were calculated as about 25%, 8%, and 2% of the total amount of putrescine, spermidine, and spermine, respectively. The method was then used to measure ultra-filtrable and macromolecule-bound polyamines in regenerating rat liver homogenates, to examine possible changes of polyamines during cell growth. The method was also applied to measure other ultra-filtrable compounds such as amino acids and inorganic ions in rat liver homogenate.

Two distinct pathways leading to nuclear apoptosis.

Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.

Regulation of both apoptosis and cell survival by the v-Src oncoprotein.

A number of oncogenes alter the regulation of the cell cycle and cell death, contributing to the altered growth of tumours. Expression of the v-Src oncoprotein in Rat-1 fibroblasts prevented cell cycle exit in response to growth factor withdrawal. Here we investigated whether survival of v-Src transformed cells in low serum is dependent on v-Src activity. We used a temperature sensitive v-Src to study the effect inactivating v-Src on transformed cells growing under low serum conditions. We found when we switched off v-Src the cells died by apoptosis characterised by activation of caspases and the stress-activated kinases, JNK (Jun N-terminal kinase) and p38 MAP (mitogen activated protein) kinase. We were able to prevent cell death by addition of serum or overexpression of Bcl-2. Thus v-Src transformed Rat-1 cells can be protected from apoptosis by serum, v-Src, or Bcl-2. We investigated how v-Src protects from apoptosis under these conditions. Amongst other effects, v-Src activates two kinases which have been shown to protect cells from apoptosis, phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK1/2). We found that switching off v-Src led to a decrease in the activity of both PI3-K and ERK1/2, however, we found that adding a specific inhibitor of PI3-K (LY294002) to v-Src transformed Rat-1 cells grown in low serum induced apoptosis while a specific ERK kinase (MEK1) inhibitor (PD98059) had no effect. This suggests that v-Src protects from apoptosis under low serum conditions by activating PI3-K.

DNA topoisomerase IIalpha interacts with CAD nuclease and is involved in chromatin condensation during apoptotic execution.

Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As chromatin condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic chromatin condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.

Detection of DNA cleavage in apoptotic cells.

At least two discrete deoxyribonuclease activities can be detected during apoptotic death, one that generates 30- to 500-kilobase pair (kbp) domain-sized fragments and another that mediates internucleosomal DNA degradation. The latter nuclease has been identified as the caspase-activated deoxyribonuclease (CAD)/CPAN, a unique enzyme that is normally inhibited by the regulatory subunit ICAD (inhibitor of CAD)/DFF45 (DNA fragmentation factor). In this chapter, techniques widely used to detect DNA cleavage in apoptotic cells, including pulsed-field gel electrophoresis, conventional agarose gel electrophoresis, and terminal transferase-mediated dUTP nick end-labeling (TUNEL), are briefly reviewed. In addition, the use of ICAD to inhibit apoptosis-associated nuclease activity is illustrated. When properly applied, these techniques are widely applicable to the characterization of apoptotic cells.

Formation of spindle poles by dynein/dynactin-dependent transport of NuMA.

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.

Differential localization of ICAD-L and ICAD-S in cells due to removal of a C-terminal NLS from ICAD-L by alternative splicing.

CAD/CPAN/DFF40 is an apoptotic nuclease that is associated with the regulatory subunit ICAD/DFF in healthy cells. ICAD has two forms, ICAD-L/DFF45 and ICAD-S/DFF35, which are transcribed from a single gene by alternative splicing. They differ at the C-terminus: 70 amino acids of ICAD-L are replaced by 4 different amino acids in ICAD-S. We previously showed that both transfected and endogenous ICAD-L are nuclear; however, the localization of ICAD and CAD remains controversial and an important issue to clarify. Here we present the evidence that ICAD-L is nuclear due to the presence of an autonomous nuclear localization signal located in the C-terminal 20 amino acids. This NLS is missing from ICAD-S, which is distributed throughout the cell. We also showed that a GFP:CAD fusion protein is located in the nucleus of transfected cells.

Probing the interaction between N(1),N(4)-dibenzylputrescine and tRNA through (15)N NMR: biological implications.

NMR spectroscopy was used to characterize the binding properties of polyamines to Escherichia coli tRNA. The (15)N NMR spectra of three (15)N-enriched N-substituted putrescine derivatives (DMP, DEP and DBP) were recorded in the presence of tRNA, and the spin relaxation times of the nitrogen nuclei were measured. From these data, the activation parameters for the rotational correlation times of the (15)N nuclei were determined. The present data indicate that the nature of the amino substituents does play a relevant role in controlling the polyamine-tRNA interaction. This study also provides a rationale for the in vivo antiproliferative effect of DBP against tumoral cells.

Role of factors downstream of caspases in nuclear disassembly during apoptotic execution.

We used cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway to analyse the events of apoptotic execution. So-called S/M extracts from morphologically normal 'committed-stage' cells induce apoptotic morphology and DNA cleavage in substrate nuclei. These apoptotic changes appear to require the function of multiple caspases (cysteine aspartases, a specialized class of proteases) acting in parallel. Extracts from 'execution-stage' apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. 'Execution-stage' extracts contain an ICAD/DFF45-inhibitable nuclease resembling CAD, plus another activity that is required for the apoptotic chromatin condensation. 'Committed-stage' S/M extracts lack these downstream activities. These observations reveal that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves. They also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to the execution phase of apoptosis.

Primary structure of rat spermidine synthase: an example of refining the cDNA-derived amino acid sequence.

The primary structure of rat spermidine synthase having the N-terminal acetylated methionine and 98.7% homology with that of the mouse enzyme is presented using a limited amount of the homogeneous enzyme. The study strategy was principally to compare the molecular masses of liberated peptides determined by three specific cleavage methods with those expected from known cDNA-derived amino acid sequences of mouse and human enzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The cleavage methods involved two enzymatic methods using lysylendopeptidase and arginylendopeptidase, and a chemical method for cleaving at the cysteine residue using 2-nitro-5-thiocyanobenzoic acid. Their usefulness was clearly demonstrated. Column-switching semimicro reversed-phase HPLC, which permits application of the entire reaction mixture, was useful for collecting a small amount of peptides containing the N-terminal amino acid, to confirm acetylation of the N-terminal methionine by MALDI TOF-MS. It was necessary in this approach to examine the amino acid sequence of certain peptides. The Edman method was used for the sequence analysis, and this will be replaced by an improved MALDI TOF-MS now available in a few laboratories.

Arginyl-tRNA-protein transferase activities in crude supernatants of rat tissues.

A fluorescent HPLC method for the assay of arginyl-tRNA-protein transferase (R-Transferase) activity was applied to obtain quantitative data of the enzyme activity in rat tissues for the first time. In this assay, the major problem was a significant hydrolysis of the substrate, N-aspartyl-N'-dansylamido-1,4-butanediamine, and the product, N-arginylaspartyl-N'-dansylamido-1,4-butanediamine (ArgAsp(4)DNS) by aminopeptidases in crude samples such as 105000g supernatants (105S) of tissue homogenates. As bestatin inhibited the hydrolysis of ArgAsp(4)DNS, a standard-addition method in the presence of bestatin, using a partially purified R-Transferase preparation from hog kidney as a standard, made it possible to measure directly R-Transferase activities in 105S with a short incubation time and sufficient reliability. It was found by the established method that of 14 tissues examined, stomach was rich in the R-Transferase activity with the highest specific activity, suggesting a target tissue for the future studies on R-Transferase to elucidate its physiological significance.