Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance.

OBJECTIVE: Notch signaling has been identified as a critical regulator in cartilage development and joint maintenance, and loss of Notch signaling in all joint tissues results in an early and progressive osteoarthritis (OA)-like pathology. This study investigated the targeted cell population within the knee joint in which Notch signaling is required for normal cartilage and joint integrity. METHODS: Two loss-of-function mouse models were generated with tissue-specific knockout of the core Notch signaling component, RBPjκ. The AcanCre(ERT2) transgene specifically removed Rbpjκ floxed alleles in postnatal joint chondrocytes, while the Col1Cre(2.3kb) transgene deleted Rbpjκ in osteoblast populations, including subchondral osteoblasts. Mutant and control mice were analyzed via histology, immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR), X-ray, and microCT imaging at multiple time-points. RESULTS: Loss of Notch signaling in postnatal joint chondrocytes results in a progressive OA-like pathology, and triggered the recruitment of non-targeted fibrotic cells into the articular cartilage potentially due to mis-regulated chemokine expression from within the cartilage. Upon recruitment, these fibrotic cells produced degenerative enzymes that may lead to the observed cartilage degradation and contribute to a significant portion of the age-related OA-like pathology. On the contrary, loss of Notch signaling in subchondral osteoblasts did not affect normal cartilage development or joint maintenance. CONCLUSIONS: RBPjκ-dependent Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance.

An integrated physical map of 8q22-q24: use in positional cloning and deletion analysis of Langer-Giedion syndrome.

We have developed an integrated map for a 35-cM area of human chromosome 8 surrounding the Langer-Giedion syndrome deletion region. This map spans from approximately 8q22 to 8q24 and includes 10 hybrid cell intervals, 89 polymorphic STSs, 118 ESTs, and 37 known genes or inferred gene homologies. The map locations of 25 genes including osteoprotegerin, syndecan-2, and autotaxin have been refined from the general locations previously reported. In addition, the map has been used to indicate the location of nine deletions in patients with Langer-Giedion syndrome and trichorhinophalangeal syndrome type I to demonstrate the potential usefulness of the map in the analysis of these complex syndromes. The map will also be of interest to anyone trying to clone positionally disease genes in this region, such as Cohen syndrome (8q22-q23), Klip-Feil syndrome (8q22.2), hereditary spastic paraplegia (8q24), and benign adult familial myoclonic epilepsy (8q23.3-q24.1).

Herpes simplex virus induces intracellular redistribution of E2F4 and accumulation of E2F pocket protein complexes.

Accumulation of E2F-p107 and E2F-pRB DNA binding complexes occurred after herpes simplex virus infection of U2-OS cells. Accumulation of E2F-p107 also occurred by 4 h p.i. in C33 cells. This corresponded to a time when host DNA synthesis was reduced by 50%, and lagged by >/=1 h, the onset of viral DNA synthesis. To determine the basis for increased nuclear E2F complexes, we investigated the effects of virus infection on the intracellular distribution of the E2F-dependent DNA binding complexes and their protein constituents. Western blot analyses of whole cell extracts revealed that amounts of E2F4, E2F1, DP1, and p107 remained unchanged after infection of C33 cells. Analysis of cytoplasmic and nuclear fractions, however, revealed that cytoplasmic E2F4 decreased and nuclear E2F4 increased. This correlated with a loss of cytoplasmic E2F DNA-binding activity and a corresponding increase in nuclear DNA-binding activity. Concomitant with its redistribution, the apparent molecular weight of total and p107-associated E2F4 increased, at least partially as a result of protein phosphorylation. Increased nuclear E2F-pRB in U2-OS cells was accompanied by the conversion of pRB from a hyper- to a hypophosphorylated state. Infection of U2-OS cells with viral mutants indicated that viral protein IE ICP4 was necessary for the decrease in cytoplasmic E2F-p107, and that viral protein DE ICP8 was required for nuclear accumulation of p107-E2F. In contrast, ICP8 was not required for accumulation of E2F-pRB. These results indicate that the increase in E2F-p107 may be explained by the redistribution and modification of E2F4 and the increase in E2F-pRB by modification of pRB.

An inhibitor of the sodium pump obtained from human placenta.

BACKGROUND: Much effort has been expended in the search for an endogenous inhibitor of the cellular sodium/potassium pump, a compound of major physiological importance, which has been implicated in the mechanism of essential hypertension. Others have suggested that ouabain or an isomer of ouabain may be the endogenous pump inhibitor. Neonatal cord serum contains an inhibitor of the sodium pump; we attempted to isolate and characterise this substance from human placentas. METHODS: Homogenised placentas were dialysed and the resulting solutes were trapped on octadecylsilyl silica and then separated by high-performance liquid chromatography. Measurement of the activity of the sodium pump of human leucocytes was used to test each fraction for the presence of the inhibitor. FINDINGS: An inhibitor of the sodium pump was obtained by this technique in a mass spectrometrically pure form with a mass of 370 Da, an empirical formula of C24H34O3 and only one hydroxyl group. The characteristic fragmentation pattern observed in negative-ion mass spectrometry was compared with those of various model compounds; this comparison suggested that the active material was a dihydropyrone-substituted steroid. INTERPRETATION: These results suggest that a dihydropyrone-substituted steroid is an endogenous regulator of the sodium pump in humans and, presumably, other mammals. Proof of the endogenous origin will require the demonstration of a previously unrecognised biosynthetic pathway.

Induction by herpes simplex virus of free and heteromeric forms of E2F transcription factor.

We have determined that HSV causes rapid and large increases in cell-cycle-regulated free E2F and S-phase p107/E2F DNA binding activities in asynchronous cultures of C33A cells. Induction occurred by 4 hr postinfection and coincided with the appearance of viral encoded immediate-early and delayed-early proteins, i.e., when viral DNA replication normally commences. No increase in E2F activities occurred when cells were infected with viruses expressing mutant regulatory proteins ICP4 or ICP27, or mutant replication proteins ICP8, pol or helicase, or when cells were infected with wild-type virus in the presence of inhibitors of DNA synthesis. In contrast, ICP8 mutant-infected cells contained elevated amounts of NF kappa B activity equivalent to WT virus, no induction of Sp1 relative to WT virus, and reduced ATF/CREB activity relative to WT virus. Results of transient expression assays with E2F-responsive reporters indicated that the net effect of induction of both active (free E2F) and repressive (p107/E2F) complexes was a decrease in AdE2 promoter activity and an increase in c-myc promoter activity. Taken together these results suggest that HSV can cause unscheduled changes in the amount and functional status of a cell-cycle-regulated transcription factor. These results are discussed in light of possible roles for viral-induced alterations in E2F, especially as related to imposing or overriding cell-cycle checkpoints.