Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.     

Hollow-fiber membrane bioreactors using immobilized E. coli for protein synthesis

Actively growing Escherichia coli C600(pBR322), immobilized within the macroporous matrix of asymmetric-wall hollow-fiber membranes, has been propagated to extremely high densities, typically more than 10(12) cells/mL of accessible void volume, in some regions cells accounting for nearly 100% of the available macrovoid volume forming a tissue-like mass. Production rates of beta-lactamase, an enzyme used as an indicator of the culture's biosynthetic potential, remained at high and relatively stable levels for more than three weeks of continuous operation, and effluent supernatant enzyme activities attained 25% of the accumulated level measured in a 24-h shaker-flask culture. Based on the accessible void volume within the fiber wall, the beta-lactamase productivity was independent of the specific asymmetric membrane used. On a per cell basis, however, cells cultured using hollow-fiber membranes were only 10% as productive as those in the shaker-flask culture, possibly due to the high packing density or culture aging. By contrast, the hollow-fiber bioreactor was 100 times more productive than the shaker-flask culture on a reactor-volume basis, primarily as a consequence of the high cell densities. Reactor productivity was dependent on the number of cells in the reactor, suggesting that reactor performance was kinetically controlled and not mass transport limited.     

Inhibition of PARP activation by enalapril is crucial for its renoprotective effect in cisplatin-induced nephrotoxicity in rats

Oxidative stress-induced PARP activation has been recognized to be a main factor in the pathogenesis of cisplatin-induced nephrotoxicity. Accumulating literature has revealed that ACE inhibitors may exert beneficial effect in several disease models via preventing PARP activation. Based on this hypothesis, we have evaluated the renoprotective effect of enalapril, an ACE inhibitor, and its underlying mechanism(s) in cisplatin-induced renal injury in rats. Male Albino Wistar rats were orally administered normal saline or enalapril (10, 20 and 40?mg/kg) for 10 days. Nephrotoxicity was induced by a single dose of cisplatin (8?mg/kg; i.p.) on the 7th day. The animals were thereafter sacrificed on the 11th day and both the kidneys were excised and processed for biochemical, histopathological, molecular, and immunohistochemical studies. Enalapril (40?mg/kg) significantly prevented cisplatin-induced renal dysfunction. In comparison to cisplatin-treated group, the elevation of BUN and creatinine levels was significantly less in this group. This improvement in kidney injury markers was well substantiated with reduced PARP expression along with phosphorylation of MAPKs including JNK/ERK/p38. Enalapril, in a dose-dependent fashion, attenuated cisplatin-induced oxidative stress as evidenced by augmented GSH, SOD and catalase activities, reduced TBARS and oxidative DNA damage (8-OHDG), and Nox-4 protein expression. Moreover, enalapril dose dependently inhibited cisplatin-induced inflammation (NF-κB/IKK-β/IL-6/Cox-2/TNF-α expressions), apoptosis (increased Bcl-2 and reduced p53, cytochrome c, Bax and caspase-3 expressions, and TUNEL/DAPI positivity) and preserved the structural integrity of the kidney. Thus, enalapril attenuated cisplatin-induced renal injury via inhibiting PARP activation and subsequent MAPKs/TNF-α/NF-κB mediated inflammatory and apoptotic response.     

Genetic variation in TIMP1 but not MMPs predict excess FEV1 decline in two general population-based cohorts

Background

An imbalance in Matrix MetalloProteases (MMPs) and Tissue Inhibitors of MMPs (TIMPs) contributes to Chronic Obstructive Pulmonary Disease (COPD) development. Longitudinal studies investigating Single Nucleotide Polymorphisms (SNPs) in MMPs and TIMPs with respect to COPD development and lung function decline in the general population are lacking.

Methods

We genotyped SNPs in MMP1 (G-1607GG), MMP2 (-1306 C/T), MMP9 (3 tagging SNPs), MMP12 (A-82G and Asn357Ser) and TIMP1 (Phe124Phe and Ile158Ile) in 1390 Caucasians with multiple FEV₁ measurements from a prospective cohort study in the general population. FEV₁ decline was analyzed using linear mixed effect models adjusted for confounders. Analyses of the X-chromosomal TIMP1 gene were stratified according to sex. All significant associations were repeated in an independent general population cohort (n = 1152).

Results

MMP2 -1306 TT genotype carriers had excess FEV₁ decline (-4.0 ml/yr, p = 0.03) compared to wild type carriers. TIMP1 Ile158Ile predicted significant excess FEV₁ decline in both males and females. TIMP1 Phe124Phe predicted significant excess FEV₁ decline in males only, which was replicated (p = 0.10) in the second cohort. The MMP2 and TIMP1 Ile158Ile associations were not replicated. Although power was limited, we did not find associations with COPD development.

Conclusions

We for the first time show that TIMP1 Phe124Phe contributes to excess FEV₁ decline in two independent prospective cohorts, albeit not quite reaching conventional statistical significance in the replication cohort. SNPs in MMPs evidently do not contribute to FEV₁ decline in the general population.     

Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling

MicroRNAs (miRNAs) play critical roles in a variety of biological processes in diverse organisms, including mammals. In the mouse skeletal system, a global reduction of miRNAs in chondrocytes causes a lethal skeletal dysplasia. However, little is known about the physiological roles of individual miRNAs in chondrocytes. The miRNA-encoding gene, Mir140, is evolutionarily conserved among vertebrates and is abundantly and almost exclusively expressed in chondrocytes. In this paper, we show that loss of Mir140 in mice causes growth defects of endochondral bones, resulting in dwarfism and craniofacial deformities. Endochondral bone development is mildly advanced due to accelerated hypertrophic differentiation of chondrocytes in Mir140-null mice. Comparison of profiles of RNA associated with Argonaute 2 (Ago2) between wild-type and Mir140-null chondrocytes identified Dnpep as a Mir140 target. As expected, Dnpep expression was increased in Mir140-null chondrocytes. Dnpep overexpression showed a mild antagonistic effect on bone morphogenetic protein (BMP) signaling at a position downstream of Smad activation. Mir140-null chondrocytes showed lower-than-normal basal BMP signaling, which was reversed by Dnpep knockdown. These results demonstrate that Mir140 is essential for normal endochondral bone development and suggest that the reduced BMP signaling caused by Dnpep upregulation plays a causal role in the skeletal defects of Mir140-null mice.     

Establishment and maintenance of nuclear position during zoospore formation in allomyces macrogynus: roles of the cytoskeleton

The involvement of the microtubule (MT) and actin microfilament (MF) cytoskeletons in establishing nuclear positions during zoosporogenesis in Allomyces macrogynus was assessed using selective cytoskeletal disrupting treatments and documented with light microscopy. These experiments were coupled with low-speed centrifugation studies to determine the degree to which cytoskeletal elements anchor nuclear position. At the onset of zoospore formation, nuclei were positioned only in cortical cytoplasmic regions of the zoosporangia (ZS). Immunofluorescence microscopy revealed that MTs primarily emanated from centrosomal regions into the surrounding cytoplasm at this stage. During delimitation of the cytoplasm into individual uninucleate zoospores, nuclei migrated from cortical regions to become distributed throughout the cytoplasm. Coincident with nuclear migrations, MTs were primarily organized at and emanated from nuclear surfaces, forming extensive perinuclear arrays. Nuclear migrations were suppressed in ZS induced to sporulate in the presence of cytochalasin D, an actin MF inhibiting compound. Disruption of MTs with nocodazole did not block nuclear migrations, although resultant nuclear spacing was irregular. Centrifugation treatments of control and drug-treated ZS demonstrated that nuclear positions were stabilized by perinuclear MT arrays. The results indicate that nuclear motility in ZS of A. macrogynus is the result of an actin-based system while perinuclear MTs arrays function to establish and fix nuclear position during zoospore formation. Copyright 1998 Academic Press.     

The National QAAMS Program – A Practical Example of PoCT Working in the Community

The Quality Assurance for Aboriginal and Torres Strait Islander Medical Services (QAAMS) Program is the largest and longest-standing national point-of-care testing (PoCT) program in Australia. With a focus on PoCT for diabetes management, it now operates in 115 Indigenous medical services and has been funded continuously by the Australian Government for 11 years. A recent independent evaluation of the QAAMS Program concluded that the program continues to meet best practice standards for Indigenous healthcare, diabetes management and PoCT.