Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma.

Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into severe combined immunodeficient (SCID) or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism-activation of p38 signaling in myeloma cells-by which myeloma cells induce osteolytic bone lesions, and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease.

Ability of induced corpora lutea to maintain pregnancy from the third month of gestation to term in cattle.

The local relationship between the pregnant uterine horn and the CL during maternal recognition of pregnancy is well-documented. It continues beyond that time; pregnancies were maintained in lutectomized cows when CL were induced on the ovary ipsilateral, but not contralateral, to the uterine horn of pregnancy during Days 28-53. This study evaluated factors affecting maintenance of pregnancy by CL induced after Day 53, in lutectomized cows that had received exogenous progesterone from Day 29 to 15 days after induction of a CL. Twenty-four suckled beef cows were lutectomized on Day 29 of gestation; pregnancy was maintained with progesterone from two controlled internal drug releasing (CIDR) inserts, exchanged every 5 days. Beginning on Day 53, ovaries and viability of pregnancy were evaluated by ultrasonography every 5 days. When a follicle >or=10 mm in diameter was present ipsilateral to the fetus, each cow received 1,000 IU of hCG. Following induction of a CL (20 of 24), progesterone was reduced to a single CIDR for 5 days, then removed. Retention of pregnancy was confirmed by rectal palpation and calving. Cows with induced CL maintained pregnancy to term, including four with the CL contralateral to the fetus. Three cows failed to form normal CL by Day 98 and lost pregnancy after removal of exogenous progesterone. One cow that did not respond to hCG lost pregnancy during exogenous progesterone. In conclusion, CL induced after Day 53 maintained pregnancy to term, even when induced contralateral to the pregnant uterine horn.

Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes.

Type I collagen is composed of two chains, alpha1(I) and alpha2(I), encoded by two distinct genes, the alpha1(I) and alpha2(I) collagen genes, that are highly expressed in osteoblasts. In most physiological situations, alpha1(I) and alpha2(I) collagen expression is coregulated, suggesting that identical transcription factors control their expression. Here, we studied the role of Cbfa1, an osteoblast-specific transcription factor, in the control of alpha1(I) and alpha2(I) collagen expression in osteoblasts. A consensus Cbfa1-binding site, termed OSE2, is present at the same location in the alpha1(I) collagen promoter at approximately -1347 base pairs (bp) of the rat, mouse, and human genes. Cbfa1 can bind to this site, as demonstrated by electrophoretic mobility shift assay (EMSA) and supershift experiments using an anti-Cbfa1 antibody. Mutagenesis of the alpha1(I) collagen OSE2 at -1347 bp reduced the activity of a alpha1(I) collagen promoter fragment 2- to 3-fold. Moreover, multimers of this OSE2 at -1347bp confer osteoblast-specific activity to a minimum alpha1(I) collagen promoter fragment in DNA transfection experiments as well as in transgenic mice. An additional Cbfa1-binding element is present in the alpha1(I) collagen promoter of mouse, rat, and human at approximately position -372. This site binds Cbfa1 only weakly and does not act as a cis-acting activator of transcription when tested in DNA transfection experiments. Similar to alpha1(I) collagen, the mouse alpha2(I) collagen gene contains multiple OSE2 sites, of which one is conserved across multiple species. In EMSA, Cbfa1 binds to this site and multimers of this alpha2(I) OSE2 element confer osteoblast-specific activity to the minimum alpha1(I) collagen promoter in DNA transfection experiments. Thus, our results suggest that Cbfa1 is one of the positive regulators of the osteoblast-specific expression of both type I collagen genes.

Broadly altered expression of the mRNA isoforms of FE65, a facilitator of beta amyloidogenesis, in Alzheimer cerebellum and other brain regions.

FE65 is a key "adapter" protein that links a multiprotein complex to an intracellular domain of beta-amyloid precursor protein (betaPP). Its overexpression modulates the trafficking of betaPP and facilitates the generation of beta-amyloid (Abeta). FE65 is predominantly expressed in brain tissues. An exon 9-inclusive isoform is exclusively expressed in neurons, and an exon 9-exclusive isoform is only expressed in non-neuronal cells. We quantitated the two isoforms in middle temporal cortex, middle frontal cortex, cerebellar cortex and caudate nucleus of 17 Alzheimer disease (AD) patients, 12 normal controls and 9 non-AD neurodegenerative disease controls by reverse transcription-competitive polymerase chain reaction (RT-cPCR). Expression of the two isoforms was significantly and differentially altered, with a 30-57% decrease in levels of the neuronal form (P < 0.05-0.002) and a 73-135% increase in levels of non-neuronal form (P < 0.02-0.001), in the temporal and frontal cortex of AD brains. These alterations presumably reflect advanced neurodegenerative processes of these regions. Surprisingly, expression of both isoforms was significantly up-regulated by 42-66% in the cerebellar cortex and caudate nucleus of AD brains when compared to normal brains (P < 0.05-0.005). Diffuse Abeta-positive plaques were observed in the cerebellum of these AD subjects but not in the normal controls. Selective up-regulation of only the FE65 neuronal isoform was seen in the cerebellar cortex in association with other neurodegenerative diseases (largely Parkinson's disease). Because FE65 modulates trafficking of betaPP toward the production of Abeta, the up-regulation of FE65 in AD cerebellum may be relevant to the genesis of diffuse plaques. Thus, early biochemical alterations in AD, not complicated by advanced pathology, may be beneficially investigated in the less-affected regions of the brain, such as the cerebellum.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR).

The polymerase chain reaction (PCR) has been used to detect Listeria monocytogenes in whole milk at a level of 0.1 cfu per 30 ml. This high degree of sensitivity has been achieved following enzymatic digestion, polysulphonone membrane filtration and amplification of a nucleotide sequence within the promoter region of hlyA. Key elements of the procedure are the absence of enrichment culture and a complete solubilization of the membrane filter, ensuring total nucleic acid recovery. The simplicity of the protocol coupled with high sample volumes and exquisite sensitivity extends the relevance of PCR within food and environmental microbiology.

Avian sarcoma virus 17 carries the jun oncogene.

Biologically active molecular clones of avian sarcoma virus 17 (ASV 17) contain a replication-defective proviral genome of 3.5 kilobases (kb). The genome retains partial gag and env sequences, which flank a cell-derived putative oncogene of 0.93 kb, termed jun. The jun gene lacks preserved coding domains of tyrosine-specific protein kinases. It also shows no significant nucleic acid homology with other known oncogenes. The probable transformation-specific protein in ASV 17-transformed cells is a 55-kDa gag-jun fusion product.

Ethylene control of anthocyanin synthesis in sorghum.

Light-induced anthocyanin synthesis in Sorghum vulgare L. seedlings was both promoted and inhibited by ethylene treatment. The rate of anthocyanin formation in sorghum tissue was dependent upon the time of ethylene treatment in relation to light exposure and the stage of the anthocyanin synthesis process. Those plants receiving ethylene treatment during the early lag phase of anthocyanin synthesis had higher anthocyanin content at 24 hours than control plants receiving no ethylene treatment. Plants receiving ethylene treatment after the lag phase had lower anthocyanin content at 24 hours than control plants receiving no ethylene treatment.