Characterization of spontaneous malignant lymphomas in Japanese macaques (Macaca fuscata).

Lymphomas are common spontaneous tumors in nonhuman primates but remain poorly characterized in Japanese macaques (Macaca fuscata). This study examined 5 cases of spontaneous malignant lymphoma in Japanese macaques, focusing on the immunophenotypes and presence of simian lymphocryptoviruses, which are Epstein-Barr virus-related herpesviruses in nonhuman primates. The macaques with lymphoma were 5 to 28 years old, indicating that lymphomas develop over a wide age range. The common macroscopic findings were splenomegaly and enlargement of lymph nodes. Histologic and immunohistochemical analyses revealed that all cases were non-Hodgkin type and exhibited a T-cell phenotype, positive for CD3 but negative for CD20 and CD79α. The lymphomas exhibited diverse cellular morphologies and were subdivided into 3 types according to the World Health Organization classification. These included 3 cases of peripheral T-cell lymphoma, not otherwise specified; 1 case of T-cell prolymphocytic leukemia; and 1 case of an unclassifiable T-cell lymphoma. Positive signals were detected by in situ hybridization in 2 of the 4 examined cases using probes for the Epstein-Barr virus-encoded small RNA (EBER). Furthermore, the presence of M. fuscata lymphocryptovirus 2, a macaque homolog of Epstein-Barr virus, was demonstrated in EBER-positive cases by polymerase chain reaction amplification followed by direct sequencing. Immunohistochemistry using antibody to the Epstein-Barr virus-encoded nuclear antigen 2 was negative, even in the EBER-positive cases. The present study suggests that T-cell lymphoma is more common than B-cell lymphoma in Japanese macaques and that M. fuscata lymphocryptovirus 2 is present in some cases.

The effect of posture on Cheyne-Stokes respirations and hemodynamics in patients with heart failure.

STUDY OBJECTIVES: Cheyne-Stokes respirations occur in 40% of patients with heart failure. Orthopnea is a cardinal symptom of heart failure and may affect the patient's sleeping angle. The objective of this study was to assess the respiratory and hemodynamic response to sleeping angle in a group of subjects with stable heart failure. DESIGN: Twenty-five patients underwent overnight polysomnography with simultaneous and continuous impedance cardiographic monitoring. Sleeping polysomnographic and impedance cardiographic data were recorded. SETTING: The study was conducted in a sleep center. PATIENTS: All 25 patients had clinically stable heart failure and left ventricular ejection fractions < 40%. INTERVENTIONS: The patients slept at 0 degrees, 15 degrees, 30 degrees, and 45 degrees in random order. MEASUREMENTS AND RESULTS: Seventeen patients had Cheyne-Stokes apneas (index > 5/h) and 23 patients had hypopneas (index > 5/h). The hypopnea index showed no response to sleeping angle. The Cheyne-Stokes apnea index decreased with increasing sleeping angle (P < 0.001). This effect was seen only during supine sleep and non-rapid eye movement sleep and was absent in non-supine sleep, rapid eye movement sleep, and during periods of wakefulness. Thoracic fluid content index and left ventricular hemodynamics measured by impedance cardiography showed no response to sleeping angle. CONCLUSIONS: Changing the heart failure patient's sleeping angle from 0 degrees to 45 degrees results in a significant decrease in Cheyne-Stokes apneas. This decrease occurs on a constant base of hypopneas. The changes in Cheyne-Stokes apneas are not related to changes in lung congestion and left ventricular hemodynamics.

Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta).

Multifocal submucosal stromal tumors were diagnosed in a 5.5-year-old rhesus macaque (Macaca mulatta) experimentally infected with simian immunodeficiency virus, strain SIVsmE660, and CD4+ T cell depleted. The animal was negative for simian retroviruses, SRV-1, -2, and -5. Polymerase chain reaction analysis of DNA from tumor and spleen tissue revealed abundant, preferential presence of retroperitoneal fibromatosis herpesvirus, the macaque homologue of the Kaposi sarcoma-associated herpesvirus (human herpesvirus-8), in the tumors. This was corroborated by demonstration of viral latent nuclear antigen-1 in the nuclei of a majority of the spindeloid tumor cells. Low levels of an additional macaque herpesvirus, rhesus rhadinovirus, were also detected in the spleen and tumor tissues. The spindeloid cells labeled positively for vimentin and CD117 but were negative for CD31, CD68, desmin, and smooth muscle cell actin. Collectively, these findings suggest a relation to but not absolute identity with simian mesenchymoproliferative disorders (MPD) or typical gastrointestinal stromal tumors (GISTs).

Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication.

Individuals with the recessive condition trimethylaminuria exhibit variation in metabolic detoxication of xenobiotics by hepatic flavin-containing monooxygenases. We show here that mutations in the human flavin-containing monooxygenase isoform 3 gene ( FMO3 ) impair N -oxygenation of xenobiotics and are responsible for the trimethylaminuria phenotype. Three disease-causing mutations in nine Australian-born probands have been identified which share a particular polymorphic haplotype. Nonsense and missense mutations are associated with a severe phenotype and are also implicated in impaired metabolism of other nitrogen- and sulfur-containing substrates including biogenic amines, both clinically and when mutated proteins expressed from cDNA are studied in vitro . These findings illustrate the critical role played by human FMO3 in the metabolism of xenobiotic substrates and endogenous amines.

CNI-H0294, a nuclear importation inhibitor of the human immunodeficiency virus type 1 genome, abrogates virus replication in infected activated peripheral blood mononuclear cells.

Active nuclear importation of the human immunodeficiency virus (HIV) type 1 (HIV-1) preintegration complex (PIC) is required for the productive infection of nondividing cells, but it is believed to be dispensable for the infection of proliferating cells, such as activated T lymphocytes. To investigate this question, we exploited the properties of the small arylene bis (methyl ketone) compound CNI-H0294. We have previously shown that this compound associated with the HIV-1 matrix protein nuclear localization sequence and blocked binding of the HIV-1 PIC to yeast karyopherin alpha. CNI-H0294 abrogated nuclear importation of the HIV-1 genome in macrophages and effectively inhibited infection of nondividing cells. In this study we demonstrate that CNI-H0294 inhibits binding of the HIV-1 PIC to human karyopherin alpha and reduces nuclear importation of the viral genome in primary peripheral blood mononuclear cells (PBMCs). We also demonstrate that CNI-H0294 inhibits acute infection of PBMC cultures in vitro with a primary isolate of HIV-1 and reduces virus replication and virus load in cultures of endogenously infected PBMCs from seropositive individuals. Thus, as for infection of nondividing, terminally differentiated macrophages, HIV-1 uses active nuclear importation of the virus genome to infect activated CD4+ T cells. These results support nuclear importation as a novel target and CNI-H0294 and its derivatives as novel compounds for therapeutic intervention in HIV infection and AIDS.

Mandibular reconstruction with transforming growth factor-beta1.

HYPOTHESIS: Transforming growth factor-beta1 (TGF-beta1) plus demineralized bone matrix (DBM) will reconstruct a critical mandibular defect devoid of periosteum in a canine model. STUDY DESIGN: Randomized, blinded, placebo-controlled, prospective animal pilot study. METHODS: Canine critical mandibular defects devoid of periosteum were reconstructed with DBM (group 1, n = 3) and DBM plus TGF-beta1 (250 microg TGF-beta1/g DBM) (group 2, n = 3). Radiologic, histologic, and biomechanical testing was performed on the test group and control group specimens at 12 weeks after implantation. RESULTS: A palpable bone bridge was present in the group 2 subjects 5 to 6 weeks after implantation and was never present in the group 1 subjects. Radiologic and histologic examination at the time of harvest (12 weeks after implantation) demonstrated a solid bone bridge in the group 2 subjects and a fibrous union in the group 1 subjects. Group 2 specimens demonstrated failure in four-point bending testing at an average maximum moment of 9.9 +/- 2.2 N-m. This value was 9.4% of the maximum moment of the contralateral nonoperated side. Group 1 specimens were palpably flexible on plate removal and had a biomechanical strength of 0. The difference in strength between group 1 and group 2 was statistically significant (P < 0.02), supporting the hypothesis that the addition of TGF-beta1 to the DBM carrier resulted in the formation of significantly stronger bone in the critical gap. CONCLUSION: The addition of TGF-beta1 to DBM results in healing of a critical bone defect devoid of periosteum in a higher mammalian model.

Differential expression and sequence-specific interaction of karyopherin alpha with nuclear localization sequences.

The process of nuclear protein transport requires the interaction of several different proteins, either directly or indirectly with nuclear localization or targeting sequences (NLS). Recently, a number of karyopherins alpha, or NLS-binding proteins, have been identified. We have found that the karyopherins hSRP1 and hSRP1alpha are differentially expressed in various leukocyte cell lines and could be induced in normal human peripheral blood lymphocytes. We show that the two karyopherins bind with varied specificities in a sequence specific manner to different NLSs and that the sequence specificity is modulated by other cytosolic proteins. There was a correlation between binding of karyopherins alpha to different NLSs and their ability to be imported into the nucleus. Taken together, these data provide evidence for multiple levels of control of the nuclear import process.

TGF-beta 1 forms functionally normal bone in a segmental sheep tibial diaphyseal defect.

OBJECTIVE: Transforming growth factor-beta 1 (TGF-beta 1) is a polyfunctional regulatory cytokine that has been shown to have roles in extracellular matrix interactions, soft tissue healing, and osteogenesis. This study was undertaken to determine the efficacy of TGF-beta 1 in the formation of functionally normal bone in tibial-diaphyseal defects. METHOD: Seven hundred fifty micrograms of recombinant human TGF-beta 1 was added to a guanidine-extracted demineralised bone matrix (Gu-DBM) carrier and the implants were used to fill a 2.5 cm tibial diaphyseal defect in skeletally mature female sheep. The defects were allowed to heal over a 12-week period. After sacrifice, they were analyzed using four-point bending mechanical testing. RESULTS: Implants with TGF-beta 1 showed complete bony bridging of the defect and stress-strain curves similar to the normal contralateral limb, while implants with the carrier alone failed to bridge the gap. CONCLUSIONS: These results demonstrate the ability of TGF-beta 1 to induce new bone which has structural and functional characteristics similar to normal bone.

Oncostatin-M: a new bone active cytokine that activates osteoblasts and inhibits bone resorption.

Osteoblasts and their precursors respond to specific cytokines, growth factors, and hormones. One facet of this response includes the secretion of additional cytokines, some of which are part of the circuitry involved in the regulation of osteoblast and osteoclast function. Therefore, understanding which cytokines are able to activate osteoblastic cells and the consequences of that activation are central to understanding normal and pathologic bone remodeling. Oncostatin M (OSM) is a glycoprotein belonging to a new subfamily of cytokines related by sequence and structural homology and the use of the signal transducing receptor component gp130. Osteoblastic cells secrete and respond to leukemia-inhibiting factor (LIF) both in vitro and in vivo, suggesting that LIF is an autocrine regulatory factor. OSM is closely related to LIF, and therefore we hypothesized that OSM should regulate the function of cells in the osteoblastic lineage. Primary neonatal murine or fetal rat calvarial osteoblastic cultures were treated with OSM or LIF and a series of biochemical and biological parameters were determined. In these cultures, OSM induced proliferation, collagen synthesis, and interleukin-6 secretion, whereas it inhibited alkaline phosphatase activity. Bone resorption was also inhibited by OSM. These data represent the first report of OSM's effects on bone cell function and indicate that, like some other members of the LIF/interleukin-6 subfamily, OSM has potent bone regulatory activity.

Transforming growth factor-beta 1 in a guanidine-extracted demineralized bone matrix carrier rapidly closes a rabbit critical calvarial defect.

Transforming growth factor beta 1 (TGF-beta 1) is a polyfunctional regulatory cytokine that has been shown to have roles in extracellular matrix interactions, soft tissue healing, and osteogenesis. Twenty-five microL of recombinant human TGF-beta 1 was added to guanidine-extracted demineralized bone matrix carrier and the implants were used to fill a 14-mm osteoperiosteal critical calvarial defect in New Zealand white rabbit model. The defects were allowed to heal over 4 weeks and the degree of new bone formation was assess by radiodensitometry and undecalcified bone histomorphometry techniques. Implants with TGF-beta 1 showed complete bridging of the gap with new bone in all cases, while the controls showed fibrous tissue repair of the gap with little or no new bone formation. These results demonstrate the ability of TGF-beta 1 to induce new bone in a brief time period in an inactive carrier.

Oncostatin M (OSM) inhibits the differentiation of pluripotent embryonic stem cells in vitro.

Oncostatin M (OSM) is a cytokine which shares a common gene structure and amino acid sequence similarity with leukemia inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6), suggesting evolution from a common ancestral gene. These four cytokines share several biological activities including the ability to induce the differentiation of the murine M1 myeloid leukemic cell line. To further define the functional similarities within this family, we have investigated whether OSM can substitute for LIF in the maintenance in vitro of the undifferentiated state of pluripotent embryonic stem (ES) cells. In this study, we demonstrate that human recombinant OSM is similar to LIF in its ability to inhibit the differentiation of MBL-5 murine ES cells cultured in vitro. The level of differentiation was determined by morphological criteria and by the continued expression of the embryonic stem cell-specific surface antigen defined by the ECMA-7 monoclonal antibody. Competition binding studies demonstrate that OSM binds to the LIF receptor on MBL-5 ES cells. Our results implicate OSM as a developmental regulatory factor for embryonic stem cells in vivo.

Transforming growth factor-beta accelerates osteoinduction in a craniofacial onlay model.

Recombinant human transforming growth factor beta 1 was added to a demineralized bone matrix (DBM) paste, formed into cylinders and implanted onto the cranial periosteum of New Zealand White rabbits. The TGF-beta was added at doses of 0, 0.3, 3, 30 and 75 micrograms per implant. When the implants were removed after six weeks, histomorphometric analysis of the implants showed that TGF-beta induced significantly higher levels of trabecular bone formation than in the controls (mineralized bone area 6.0 +/- 0.8, 6.0 +/- 1.2, 5.6 +/- 1.0, 10.1 +/- 1.5, and 10.8 +/- 1.4 mm2, respectively, P < 0.05), TGF-beta also caused greater resorption of the demineralized bone matrix carrier (matrix area 7.2 +/- 0.9, 6.8 +/- 1.4, 3.7 +/- 0.9, 2.7 +/- 1.2, 0.9 +/- 0.5 mm2, respectively, P < 0.02). Measurements of the osteoid demonstrated a more active bone surface and there was evidence of rapid bone remodeling. Similar results were obtained using TGF-5 beta, a new hybrid molecule. These results demonstrate the capacity of transforming growth factor beta in accelerating osteoinduction.

Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.

Oncostatin M binds the high-affinity leukemia inhibitory factor receptor.

Oncostatin M (OSM) is a glycoprotein cytokine that was recently demonstrated to be structurally and functionally related to the leukemia inhibitory factor (LIF). We have investigated the binding of each cytokine to a variety of cellular receptors including those on solid tumor lines, leukemic cells, endothelial cells, macrophages, and cells transfected with the recently cloned low-affinity LIF receptor, and to a soluble form of the LIF receptor. LIF is incapable of binding either high- or low-affinity OSM receptors, yet OSM is capable of binding the high-affinity but not the low-affinity LIF receptor. Since the presence of high-affinity LIF receptors correlates with the biological activity of LIF on a wide range of target cells, we predict that OSM should have similar effects on LIF-responsive cells.

Oncostatin M.

Oncostatin M (OSM) was initially identified as a polypeptide cytokine which inhibited the in vitro growth of cells from melanoma and other solid tumors. OSM shows significant similarities in primary amino acid sequence and predicted secondary structure to leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), granulocyte colony-stimulating factor (G-CSF), interleukin 6 (IL-6), and interleukin 11 (IL-11). Analysis of the genes encoding these proteins reveals a shared exon organization suggesting evolutionary descent from a common ancestral gene. Recent data indicates that OSM also shares a number of in vitro activities with other members of this cytokine family. The overlapping biological effects appear to be explained by the sharing of receptor subunits.

Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6.

Oncostatin M (OSM), a glycoprotein of Mr approximately 28,000 produced by activated monocyte and T-lymphocyte cell lines, was previously identified by its ability to inhibit the growth of cells from melanoma and other solid tumors. We have detected significant similarities in the primary amino acid sequences and predicted secondary structures of OSM, leukemia-inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). Analysis of the genes encoding these proteins revealed a shared exon organization, suggesting evolutionary descent from a common ancestral gene. Using a panel of DNAs from somatic cell hybrids, we have shown that OSM, like LIF, is located on human chromosome 22. We have also demonstrated that OSM has the ability to inhibit the proliferation of murine M1 myeloid leukemic cells and can induce their differentiation into macrophage-like cells, a function shared by LIF, G-CSF, and IL-6. We propose that OSM, LIF, G-CSF, and IL-6 are structurally related members of a cytokine family that have in common the ability to modulate differentiation of a variety of cell types.

Synthesis of an active hybrid growth factor (GF) in bacteria: transforming GF-alpha/vaccinia GF fusion protein.

A hybrid gene encoding for a polypeptide consisting of the first 33 N-terminal amino acid (aa) residues of transforming growth factor-alpha (TGF-alpha) and a C terminus consisting of 20 aa residues of vaccinia growth factor (VGF) was chemically synthesized and expressed as a fusion protein in Escherichia coli. The primary structure of the hybrid gene product maintained the same positioning of the three disulfide bonds found in each parent molecule thus conserving the first two loop regions of TGF-alpha and the third loop region of VGF. After cleavage with CNBr its renatured biological activity was found to be comparable to TGF-alpha and VGF with respect to binding to the epidermal growth factor receptor, stimulation of DNA synthesis and induction of anchorage-independent growth of NRK cells in the presence of TGF-beta. Thus, we suggest that similar domains can be interchanged within the same family of molecules and equivalent functionality maintained.

tRNA anticodon replacement experiments show that ribosomal frameshifting can be caused by doublet decoding.

The expression of certain normal genes requires a specific ribosomal frameshift event because the mRNA has the coding information for one protein in two different reading frames. One of several possible mechanisms for this involves recognition of a nontriplet codon by a noncognate tRNA. The AGUC-decoding Escherichia coli tRNASer3 reads a GCA alanine codon to cause a -1 frameshift. Replacement of the anticodon of tRNAPhe with the anticodon of tRNASer3 allows the constructed tRNA to cause this frameshifting. By altering the anticodon loop nucleotides at positions 33-36 in the constructed tRNAPhe molecules, the tRNA was found to recognize a 2-base codon. Instead of the usual anticodon, positions 34-36, the nucleotides in positions 34 and 35 form essential base pairs with the first two positions of the alanine codon. The uridine in position 36 is also required but not for base pairing.

Uridine-33 in yeast tRNA not essential for amber suppression.

The nucleotide at position 33 on the 5' side of the anticodon of almost all tRNAs is a uridine. Crystallographic studies of different tRNAs reveal that although the precise orientation of uridine-33 is not always the same, it connects the anticodon stacked along the 3' side of the loop with the pyrimidine-32 stacked on the 5' side of the loop. The remarkably conserved nature of uridine-33 and its unique position in the anticodon loop structure has led to suggestions that this nucleotide has an essential role in the translational mechanism. We have developed a biochemical procedure to replace nucleotides 33-35 in yeast tRNATyr with any desired sequence and used it to construct amber suppressor tRNAs having different nucleotides at position 33. As all of these synthetic amber suppressor tRNAs functioned well in eukaryotic in vitro suppression assays, we conclude that uridine-33 does not have an obligatory role in the translation mechanism.

Replacement of anticodon loop nucleotides to produce functional tRNAs: amber suppressors derived from yeast tRNAPhe.

The method of anticodon loop replacement has been used to make derivatives of yeast tRNAPhe. By constructing tRNAs with a CUA anticodon, complementary to the amber (UAG) terminator, functional amber suppressor tRNAs were produced. The activity of these tRNAs was assayed in a mammalian cell-free protein synthesizing system. The level of suppression reflects the efficiency of codon recognition. tRNAs were constructed with either A, C, U, or G on the 3' side of the CUA anticodon. The tRNAs containing the purines were efficient amber suppressors, whereas those containing pyrimidines were inefficient.