Predictive factors of hospital stay in patients with odontogenic maxillofacial infections: the role of C-reactive protein.

To investigate whether clinical or laboratory variables on admission of patients with odontogenic infections are associated with a severe clinical course and a prolonged hospital stay, we hypothesised that specific factors such as the serum concentration of C-reactive protein (CRP) may act as predictors of the duration of stay. We designed a prospective patient-oriented study that included all those treated for maxillofacial infections of odontogenic origin in the Oral and Maxillofacial Surgery Department of Northampton General Hospital between November 2013 and December 2014. A total of 71 were enrolled. We found that the concentration of CRP was a significant predictor of hospital stay (p=0.01). Its measurement on admission can predict the likely duration of stay of these patients and enable beds to be managed more efficiently.

Spectral analysis of respiratory noise in horses with upper airway disorders.

REASONS FOR PERFORMING STUDY: It has long been recognised that the production of abnormal respiratory sounds by horses during exercise is frequently associated with upper airway obstructions. Respiratory acoustic measurements have shown promise in investigation of upper airway disorders in man and, more recently, in horses with experimentally-induced obstructions. OBJECTIVES: To evaluate sounds from exercising horses with naturally occurring dynamic obstructions of the upper respiratory tract and to compare these with those from normal horses in order to determine whether different obstructions produce characteristic spectral patterns. METHODS: The audio signal, airflow and videoendoscopic images were recorded simultaneously during an incremental exercise test on a high-speed treadmill. RESULTS: Spectral analysis of the audio signal showed marked differences between control and clinically afflicted horses. Dorsal displacement of the soft palate was characterised by a narrow low frequency (20-80 Hz) peak during expiration. Horses with dynamic laryngeal collapse produced inspiratory sounds characterised by a broad band high frequency spectral component in the range 1.1-2.7 kHz. CONCLUSIONS AND POTENTIAL RELEVANCE: Spectral analysis of respiratory sounds in horses has potential as a diagnostic technique for field use especially when facilities for high-speed treadmill assessment are not practicable.

Specialized ability of thymic epithelial cells to mediate positive selection does not require expression of the steroidogenic enzyme p450scc.

Thymic epithelial cells are uniquely efficient in mediating positive selection, suggesting that in addition to providing peptide/MHC complexes for TCR ligation, they may also provide additional support for this process. Recent studies have shown that although engagement of either the TCR or glucocorticoid (GC) receptors can individually induce apoptosis in thymocytes, together these signals are mutually antagonistic. This had led to the suggestion that local GC production by thymic epithelial cells, by opposing TCR signaling for apoptosis, provides the basis of the ability of these cells to mediate thymocyte positive selection. In this paper we have examined this possibility directly and shown that highly purified cortical epithelial cells, which have the functional ability to mediate positive selection in reaggregate cultures, do not express mRNA for the key steroidogenic enzyme P405scc. Thus we conclude that the ability of thymic epithelial cells to support positive selection does not rely on their ability to produce GC. However, we find that P450scc mRNA is up-regulated in thymocytes on the initiation of positive selection, raising the possibility that any local protective effect of steroid production is mediated at the level of thymocytes themselves.

Immune-associated nucleotide-1 (IAN-1) is a thymic selection marker and defines a novel gene family conserved in plants.

Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.

The regulation and functions of cdk7.

cdk7 started its life rather anonymously as a kinase called MO15, identified during a search for cDNA's which encode protein kinases related to cdc2. For several years its function remained obscure, but during the last 18 months MO15 has revealed itself as the catalytic subunit of cdk activating kinase, associating with at least two other subunits including a new cyclin, cyclin H. MO15(cdk7) has therefore been established paradoxically as both a new member and a regulator of the cyclin dependent kinase family. New evidence now suggests that cdk7 is also involved in the processes of transcription initiation and DNA repair, associating with the general transcription factor TFIIH. The engima of cdk7 is likely to remain for a while yet, and perhaps even more surprises are in store.

Expression and activity of p40MO15, the catalytic subunit of cdk-activating kinase, during Xenopus oogenesis and embryogenesis.

Threonine 161 phosphorylation of p34cdc2 and its equivalent threonine 160 in p33cdk2 by cdk-activating kinase (CAK) is essential for the activation of these cyclin-dependent kinases. We have studied the expression and associated kinase activity of p40MO15, the catalytic subunit of CAK, during Xenopus oogenesis, meiotic maturation, and early development to understand in more detail how cdk kinases are regulated during these events. We find that p40MO15 is a stable protein with a half-life > 16 h that is accumulated during oogenesis. p40MO15 protein and its associated CAK activity are localized predominantly to the germinal vesicle; however, a small but significant proportion is found in the cytoplasm. The amount of p40MO15 detected in stage VI oocytes remains unchanged through meiotic maturation, fertilization, and early embryogenesis. Significantly, p40MO15 was found to be constitutively active during oogenesis, meiotic maturation, and the rapid mitotic cycles of early development. This suggests that regulation of p34cdc2 and p33cdk2 activity during cell cycle progression does not involve changes in the level or activity of p40MO15/CAK.

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.

The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues.

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).

CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15.

The mitotic inducer p34cdc2 requires association with a cyclin and phosphorylation on Thr161 for its activity as a protein kinase. CAK, the p34cdc2 activating kinase, was previously identified as an enzyme necessary for this activating phosphorylation. We confirm here that CAK is a protein kinase and describe its purification over 13,000-fold from Xenopus egg extracts. We further show that CAK contains a protein identical or closely related to the previously identified Xenopus MO15 gene: p40MO15 copurifies with CAK, and an antiserum to p40MO15 specifically depletes cAK activity. CAK appears to be the only protein in Xenopus egg extracts that can activate complexes of either p34cdc2 or the closely related protein kinase, p33cdk2, with either cyclin A or cyclin B. The sequence similarity between p40MO15 and p34cdc2, and the approximately 200 kDa size of CAK, suggest that p40MO15 may itself be regulated by subunit association and by protein phosphorylations.

p40MO15, a cdc2-related protein kinase involved in negative regulation of meiotic maturation of Xenopus oocytes.

The clone MO15 which codes for a 40 kd protein (p40MO15) with 40% amino acid identity to the human cdc2 protein kinase has been isolated from a Xenopus cDNA library using a synthetic oligonucleotide probe. MO15 mRNA is accumulated during oogenesis, becomes de-adenylated during meiotic maturation, and is degraded after the mid-blastula-transition stage of embryogenesis. Translation of p40MO15 is restricted to non-mature oocytes. Specific inhibition of p40MO15 synthesis in stage VI oocytes by antisense oligonucleotide depletion of MO15 mRNA increases the rate of progesterone induced H1 kinase activation and oocyte maturation. This effect can be reversed by subsequent injection of synthetic MO15 mRNA. These results suggest that p40MO15 is involved in negatively regulating meiosis.

Antisense oligodeoxyribonucleotide-directed cleavage of maternal mRNA in Xenopus oocytes and embryos.

We have investigated the effect of specific antisense oligodeoxynucleotides (oligos) on endogenous histone H4 mRNA in Xenopus oocytes, eggs and embryos. In unfertilised eggs and non-matured oocytes, one 20-mer oligo (H4-1) mediated the RNAse H-like cleavage of up to 95% of H4 mRNA (which included polysomal mRNA), and cleavage was still obtained when the size of the oligo was reduced to a 10-mer; no cleavage was observed with 6- and 8-mers. The residual uncleaved mRNA appeared to be completely inaccessible to H4-1 since a second injection caused no further cleavage. A second 20-mer (H4-2) directed against a different region of H4 mRNA was much less effective (less than 5% cleavage). In fertilised embryos, injections of H4-1 and an oligo directed against the localised Vg1 mRNA caused less cleavage than in oocytes and also showed signs of inducing localised, non-specific mRNA cleavage. However we have been able to prepare fertilised embryos devoid of Vg1 mRNA by maturing and fertilising oligo-injected oocytes in vitro.

Antisense oligonucleotide-directed cleavage of mRNA in Xenopus oocytes and eggs.

We have investigated the effect of specific antisense oligonucleotides on both exogenous and endogenous mRNAs in Xenopus oocytes and eggs. Injection of 19- or 20-mers complementary to 70-kd heat shock protein, histone H4 and vegetally localized Veg 1 coding sequences causes rapid cleavage and degradation of up to 96% of the target transcripts present in stage VI oocytes. Nuclear and cytoplasmic transcripts appear to be equally accessible to cytoplasmically injected oligonucleotide and efficient cleavage also occurs in mature oocytes and unfertilized eggs. The residual intact mRNA appears to be completely inaccessible, resisting cleavage by further addition of oligonucleotide. We confirm that antisense oligonucleotides appear to act specifically in vivo, as previously reported in vitro, by directing RNase H cleavage and destabilization of their complementary mRNA.

Transcript levels and translational control of hsp70 synthesis in Xenopus oocytes.

Until recently it was believed that Xenopus oocytes respond to heat shock by synthesizing the 70-kD heat shock protein hsp70 and that, uniquely amongst animal cell types, this response is mediated entirely at the translational level. This view has now been challenged and we present data that reevaluate the involvement of translational control in the heat shock response of Xenopus oocytes. RNase mapping shows that up to 13 pg of hsp70A and hsp70B mRNA are accumulated by fully grown oocytes in the absence of heat shock. These transcripts are retained stably during maturation, fertilization, early cleavage, and following heat shock. However, no hsp70 protein synthesis can be detected by two-dimensional polyacrylamide gel analysis of [35S]methionine-labeled proteins from completely defolliculated oocytes, either before or during heat shock. Oocytes injected with hsp70A DNA rapidly accumulate high levels of hsp70 mRNA in their cytoplasm at normal temperature. During heat shock these oocytes accumulate more transcripts, but they remain in the nucleus and cytoplasmic levels remain constant. Translation of hsp70 from these transcripts is readily detectable at non-heat shock and heat shock temperatures. We conclude that (1) "exogenous" hsp70 transcripts are efficiently translated and not masked at normal temperatures in oocytes, and (2) oocytes are able to selectively translate hsp70 mRNA during heat shock.

Isolation and characterisation of arthropod gap junctions.

Gap junctions have been isolated from the hepatopancreas of the crustacean arthropod, Nephrops norvegicus (Norway lobster). SDS-PAGE of these preparations shows two major protein bands, mol. wt. 18 000 (18 K) and mol. wt. 28 000 (28 K). The 18-K and 28-K proteins are interconvertible, cannot be distinguished by two dimensional tryptic and chymotryptic peptide mapping, and therefore appear to be different (most likely monomeric and dimeric) forms of the same protein. The protein can also aggregate to higher multimeric forms mol. wt. 38 000 (presumed trimer), and mol. wt. 52 000 (presumed tetramer). The buoyant density of the isolated gap junctions in continuous potassium iodide gradients is 1.260 g/cm. The junctions are progressively solubilized in increasing SDS concentrations, mostly between 0.1% and 0.2% SDS, and this is accompanied by the release of the 18-K and 28-K forms of the junctional protein. The Nephrops hepatopancreas 18-K junctional protein has antigenic determinants in common with the vertebrate 16-K junctional protein as shown by cross-reactivity with two different affinity purified antibody preparations. However, no detectable similarity can be seen between the major I-labelled tryptic and chymotrytpic peptides of the Nephrops hepatopancreas 18-K protein and the mouse liver 16-K protein.

Expression of interferon-alpha and interferon-beta genes in human lymphoblastoid (Namalwa) cells.

Treatment of human lymphoblastoid (Namalwa) cells with Sendai virus induced the coordinate synthesis of both IFN-alpha and IFN-beta interferon mRNAs. One sub-line of Namalwa cells (WRL) produced no IFN-beta activity, although IFN-beta mRNA was induced and was associated with polysomes. The IFN-alpha mRNA was heterogeneous, ranging in size over 1.20-1.35 X 10(3) bases, probably because of variation in the size of the transcribale DNA in the alpha-gene family. The IFN-beta mRNA was monodisperse with a size of 1.05 X 10(3) bases. The kinetics of accumulation and decay of both IFN-alpha and IFN-beta mRNAs, as assessed by hybridization with cDNA probes, were very similar to those of translatable interferon mRNA, as assessed by translation in Xenopus oocytes. Treatment of the cells with butyrate or 5'-bromodeoxyuridine increased the amount of hybridizable IFN-alpha and IFN-beta mRNA about 15-fold and 4-fold respectively, again demonstrating coordinate control of IFN-alpha and IFN-beta production.

Analysis of vertebrate gap junction protein.

A new method for the purification of gap junctions is described which depends on the extraction of cell monolayers or tissue homogenates with Triton X-100. The major band on SDS-polyacrylamide gel electrophoresis (PAGE) of junctional preparations from a variety of vertebrate sources has an apparent mol. wt. of 16,000 (16 K). Further evidence for the junctional origin of the 16 K protein is provided by the results of four different experimental approaches. (i) The junctions form a sharp band in potassium iodide density gradients at 1.195 g/cm3 and the 16 K protein is the only detectable band in fractions of this bouyant density. (ii) The junctions are progressively solubilised by increasing concentrations of SDS (in the range 0.1-0.5%) and the dissolution of the junctional structure, observed by electron microscopy, parallels the release of the 16 K protein. (iii) Glutaraldehyde fixation of intact junctions cross-links the 16 K protein. (iv) The recoverable amount of the 16 K protein correlates with known changes in gap junctional area in the regenerating weanling rat liver after partial hepatectomy and in V79 cell cultures exposed to 4beta-phorbol 12-myristate 13-acetate.

Protein synthesis in human lymphoblastoid cells (Namalwa) after treatment with butyrate and 5'-bromodeoxyuridine.

The pattern of protein synthesis has been compared in Namalwa cells following treatment with butyrate and 5'-bromodeoxyuridine (BrdUrd). Although these treatments cause a substantial increase in Sendai virus-induced interferon synthesis (up to 300-fold) we observed no comparable effect on the synthesis of other cellular proteins. Using two-dimensional gel electrophoresis we have investigated the proteins synthesised before and 8 h after Sendai virus infection of treated cells. Only 2 of the 300 most abundant cellular proteins were reproducibly affected, these always showed increased rates of synthesis in butyrate-treated cells. The most significant was a 3-4-fold enhancement in synthesis of a 35,000 molecular weight protein which we have called BEP35. On individual occasions treatment caused changes in the rates of synthesis of other proteins, these were not reproducible and involved less than 4% of the proteins investigated. None of the Sendai virus structural proteins or virus-induced cellular proteins were affected by the treatment. We conclude that butyrate and BrdUrd treatments have a relatively specific effect on the synthesis of interferon in Namalwa cells, as the majority of protein synthesis remains unaffected.