The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G1 phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A͞C intranuclear foci. Here, we examine pRB function in cells lacking lamin A͞C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna ؊/؊ cells resemble Rb ؊/؊ cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation.

GN, UGN, and STa act on the mouse kidney, in part, through a cGMP-dependent, GC-C-independent mechanism, causing significant natriuresis by renal tubular processes. UGN may have further long-term effects on the kidney by altering the expression of such transport-associated proteins as Na+/K+ ATPase and ClC-K2.

Uroguanylin and guanylin are newly discovered endogenous heatstable peptides that bind to and activate a membrane bound guanylyl cyclase signaling receptor (termed guanylyl cyclase C; GC-C). These peptides are not only found in blood but are secreted into the lumen of the intestine and effect a net secretion of electrolytes (Na + , K + , Cl -, HCO 3 -) and fluid into the intestine via a cyclic guanosine-3',5'-monophosphate (cGMP) mechanism. GC-C is also the receptor for Escherichia coli heat-stable enterotoxin (STa) and activation by STa results in a diarrheal illness. Employing mouse renal in vivo models, we have demonstrated that uroguanylin, guanylin, and STa elicit natriuretic, kaliuretic, and diuretic effects. These biological responses are time-and dose-dependent. Maximum natriuretic and kaliuretic effects are observed within 30-40 min following infusion with pharmacological doses of the peptides in a sealed-urethra mouse model. Our mouse renal clearance model confirms these results and shows significant natriuresis following a constant infusion of uroguanylin for 30 min, while the glomerular filtration rate, plasma creatinine, urine osmolality, heart rate, and blood pressure remain constant. These data suggest the peptides act through tubular transport mechanisms. Consistent with a tubular mechanism, messenger RNA-differential display PCR of kidney RNA extracted from vehicle-and uroguanylintreated mice show the message for the Na + /K + ATPase g-subunit is down-regulated. Interestingly, GC-C knockout mice (Gucy2c -/-) also exhibit significant uroguanylin-induced natriuresis and kaliuresis in vivo, suggesting the presence of an alternate receptor signaling mechanism in the kidney. Thus, uroguanylin and guanylin seem to serve as intestinal and renal natriuretic peptide-hormones influencing salt and water transport in the kidney through GC-C dependent and independent pathways. Furthermore, our recent clinical probe study has revealed a 70-fold increase in levels of urinary uroguanylin in patients with congestive heart failure. In conclusion, our studies support the concept that uroguanylin and guanylin are endogenous effector peptides involved in regulating body salt and water homeostasis.

No abstract

In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/Cassociated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase ␦, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit.DNA replication in the mammalian cell is organized into discrete nuclear foci that change in location as S phase proceeds (3,17,32). A fundamental question is whether the DNA synthesis occurring at these sites results from motion of the replication machinery along the DNA or from spooling of the DNA through fixed replication factories. Early work revealed that many eukaryotic origins spaced throughout a chromosomal region could fire synchronously, suggesting a nuclear coordination of replication initiation (13). It was later demonstrated, using autoradiography, that newly incorporated [ 3 H]thymidine is attached to fixed positions along the nuclear matrix, suggesting the presence of replication complexes through which DNA is moved (33). Furthermore, visualization of these replication foci by electron microscopy showed spreading of label into adjacent chromatin with longer pulses of [ 3 H]thymidine (12). While providing evidence for fixed sites of DNA synthesis, these findings do not directly discriminate between movement of replication machinery and that of replicating DNA.A number of studies performed with immortalized cell lines using 5-bromo-2Ј-deoxyuridine (BrdU) labeling and immunofluorescence have shown that DNA synthesis initiates in hundreds of foci distributed throughout the nucleus (3, 32). Recent studies indicate that changes in replication patterns as S phase progresses reflect assembly and disassembly of sites of nucleotide incorporation, rather than motion, lending support to the fixed-replication-site model (6,24,38). Studies of the prokaryotic organism Bacillus subtilis, which utilizes a small number of replication foci, have provided the clearest evidence for the factory replication model (23, 28). Expression of a DNA polymerase (pol)-green fluorescent protein fusion protein showed the replication machinery to remain fixed in a few discrete positions throughout S phase. Chromosomal regions were observed to colocalize with the centrally located DNA pol and migrate...

The brain of Alzheimer's disease patients contains deposits of the 39-42-amino acid (approximately 4 kDa) amyloid beta-peptide, which is derived from the beta-amyloid precursor protein. These pathological deposits have been shown to consist in part of insoluble 8- and 16-kDa aggregates of the amyloid beta-peptide. This report confirms that the amyloid beta-peptide is a substrate for tissue transglutaminase (TGase) and demonstrates that human brain preparations from Alzheimer's disease patients and control patients form cross-linked dimers from added iodinated amyloid beta-peptide. Immunohistochemical staining for TGase revealed its presence in tissue sections and isolated amyloid plaque cores obtained from brains of patients diagnosed as having Alzheimer's disease. These results provide evidence that the previously described insoluble amyloid deposits in Alzheimer's disease may involve TGase-mediated cross-linked amyloid beta-peptide polymers, and suggest a potential role for TGase in the pathogenesis of this disease.

No abstract

This protocol describes the procedure by which the OMS Atlas serially sections a FFPE block, prepares the resulting slides, and then distributes the specimens for downstream analysis.