The adherence-associated Fdp fasciclin I domain protein of the biohydrogen producer Rhodobacter sphaeroides is regulated by the global Prr pathway.

Expression of fdp, encoding a fasciclin I domain protein important for adherence in the hydrogen-producing bacterium Rhodobacter sphaeroides, was investigated under a range of conditions to gain insights into optimization of adherence for immobilization strategies suitable for H2 production. The fdp promoter was linked to a lacZ reporter and expressed in wild type and in PRRB and PRRA mutant strains of the Prr regulatory pathway. Expression was significantly negatively regulated by Prr under all conditions of aerobiosis tested including anaerobic conditions (required for H2 production), and aerobically regardless of growth phase, growth medium complexity or composition, carbon source, heat and cold shock and dark/light conditions. Negative fdp regulation by Prr was reflected in cellular levels of translated Fdp protein. Since Prr is required directly for nitrogenase expression, we propose optimization of Fdp-based adherence in R. sphaeroides for immobilized biohydrogen production by inactivation of the PrrA binding site(s) upstream of fdp.

Estimating annual generation rates of total P and total N for different land uses in Tasmania, Australia.

Water quality issues have become increasingly important to Australian catchment stakeholders. As extensive nutrient sampling and modelling expertise are often absent or unattainable, simple unit-area models like Catchment Management Support System (CMSS) remain an attractive option for informing water quality management decisions. The selection of nutrient generation rates for use in CMSS is often an arbitrary assignment based on limited literature sources or expert opinion. Using a Bayesian model to estimate nutrient generation rates for the region of Tasmania, Australia, improved the rigor of CMSS modelling and in the process highlighted that dairy pastures were the most significant contributor of total phosphorus and total nitrogen loads to Tasmanian rivers.

One hundred questions of importance to the conservation of global biological diversity.

We identified 100 scientific questions that, if answered, would have the greatest impact on conservation practice and policy. Representatives from 21 international organizations, regional sections and working groups of the Society for Conservation Biology, and 12 academics, from all continents except Antarctica, compiled 2291 questions of relevance to conservation of biological diversity worldwide. The questions were gathered from 761 individuals through workshops, email requests, and discussions. Voting by email to short-list questions, followed by a 2-day workshop, was used to derive the final list of 100 questions. Most of the final questions were derived through a process of modification and combination as the workshop progressed. The questions are divided into 12 sections: ecosystem functions and services, climate change, technological change, protected areas, ecosystem management and restoration, terrestrial ecosystems, marine ecosystems, freshwater ecosystems, species management, organizational systems and processes, societal context and change, and impacts of conservation interventions. We anticipate that these questions will help identify new directions for researchers and assist funders in directing funds.

Is there an association between blood group O and epistaxis?

INTRODUCTION: Blood group O is associated with lower expression of von Willebrand factor suggesting a relative bleeding tendency. A lower admission rate for epistaxis among Asians compared with Caucasians has also been noted, with one explanation being higher prevalence of blood group O among Caucasians. This study investigates whether blood group O is over-represented in patients admitted with epistaxis. METHODS: A retrospective study was conducted, using computerised hospital in-patient and blood bank databases to identify Caucasians admitted with epistaxis between January 2000 and December 2005 inclusive. The control group consisted of 500 consecutive patients who had a primary total hip arthroplasty and 500 consecutive patients who gave birth within the delivery suite. RESULTS: 1261 Caucasians admitted with epistaxis were identified. Among epistaxis patients, 50.44 per cent were blood group O but among the control group this was 45.10 per cent (chi-square test p = 0.008). CONCLUSION: Blood group O appears over-represented in Caucasian patients admitted with epistaxis, compared with the control population, raising the possibility that blood group O is a risk factor for epistaxis.

The effect of mechanical strain on protease production by keratinocytes.

BACKGROUND: Intact skin is under constant tension, transmitted from the underlying dermis, but when tension is lost (i.e. upon wounding) protease activity is upregulated. OBJECTIVES: To investigate the effect of mechanical strain on protease production by both normal and transformed keratinocytes in vitro. METHODS: Keratinocytes were seeded on to membranes precoated with either type I or type IV collagen. After 48 h medium was replaced with serum-free medium and mechanical strain was applied. RESULTS: Mechanical strain resulted in decreased urokinase-type plasminogen activator (uPA) production by normal human keratinocytes (P<0.05) but increased production by transformed keratinocytes (P<0.05) cultured on type I and type IV collagen. CONCLUSIONS: Differential production of uPA by normal and transformed keratinocytes is relevant in the context of normal function, wound healing and tumorigenesis.

Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression.

OBJECTIVE: Cell immortalization is considered to be a prerequisite status for carcinogenesis. Normal human ovarian surface epithelial (OSE) cells, which are thought to be the origin of most of human ovarian carcinomas, have a very limited lifespan in culture. Establishment of immortalized OSE cell lines has, in the past, required inactivation of pRb and p53 functions. However, this often leads to increased chromosome instability during prolonged culture. MATERIALS AND METHODS: In this study, we have used a retroviral infection method to overexpress human telomerase reverse transcriptase (hTERT) gene, in primary normal OSE cells, under optimized culture conditions. RESULTS: In vitro and in vivo analysis of hTERT-immortalized cell lines confirmed their normal epithelial characteristics. Gene expression profiles and functional analysis of p16(INK4A), p15(INK4B), pRb and p53 confirmed the presence of their intact functions. Our study suggests that inactivation of pRb and p53 is not necessary for OSE immortalization. Furthermore, down-regulation of p15(INK4B) in the immortalized cells may indicate a functional role for this protein in them. CONCLUSION: These immortal OSE cell lines are likely to be an important tool for studying human OSE biology and carcinogenesis.

Evidence for redundancy in cysteine biosynthesis in Rhizobium leguminosarum RL3841: analysis of a cysE gene encoding serine acetyltransferase.

A cysE gene encoding a serine acetyltransferase (SAT) potentially involved in the biosynthesis of cysteine was identified approximately 4 kb upstream of the previously described aapJQMP gene cluster that encodes an amino acid permease in Rhizobium leguminosarum strain 3841. The gene exhibits >40% identity to the family of SATs containing N-terminal extensions that have been described for other bacteria and plants. The ORF has three possible translation initiation sites which potentially encode polypeptides of 311, 277 and/or 259 amino acid residues, respectively. All three ORFs complemented the cysE mutation in an Escherichia coli cysteine auxotroph, strain JM39. Insertion of Tn5-lacZ into cysE in the genome of R. leguminosarum (strain RU632) lowered SAT activity in crude extracts by >95%. However, RU632 was not a cysteine auxotroph, which suggests that R. leguminosarum possesses some redundancy in cysteine biosynthesis. Additional copies of cysE could not be detected in the genome when the R. leguminosarum cysE gene was used as a hybridization probe. Therefore it is possible that R. leguminosarum possesses an alternative pathway for cysteine biosynthesis which avoids O-acetylserine. Strain RU632 was unaffected in its ability to nodulate Pisum sativum, and the nodules were effective for N(2) fixation (measured by C(2)H(2) reduction). Transcriptional activity of cysE was determined by measuring the beta-galactosidase arising from cysE::Tn5-lacZ fusions. Maximal levels of expression were observed during early exponential growth and were not influenced by the level of sulphur (supplied as sulphate). However, transcription was repressed by approximately twofold in ammonium-grown, as opposed to glutamate-grown, cultures. Repression by ammonium was not seen in a strain defective for ntrC.

A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis.

In normal epidermis, beta1 integrin expression is confined to the basal layer, whereas in hyperproliferative epidermis, integrins are also expressed in the suprabasal layers. Transgenic mice in which integrins are expressed suprabasally via the involucrin promoter have a sporadic psoriatic phenotype; however, the mechanism by which integrins contribute to the pathogenesis of psoriasis is unknown. We observed activation of mitogen-activated protein kinase (MAPK) in basal and suprabasal keratinocytes of human and transgenic mouse psoriatic lesions and healing mouse skin wounds, correlating in each case with suprabasal integrin expression. Phenotypically normal human and transgenic mouse epidermis did not contain activated MAPK. Transgene-positive keratinocytes produced more IL-1alpha than controls did, and keratinocyte MAPK could be activated by ligation of suprabasal integrins or treatment with IL-1alpha. Constitutive activation of MAPK increased the growth rate of human keratinocytes and delayed the onset of terminal differentiation, recreating many of the histological features of psoriatic epidermis. We propose that activation of MAPK by integrins, either directly or through increased IL-1alpha production, is responsible for epidermal hyperproliferation in psoriasis and wound healing, and that the sporadic phenotype of the transgenic mice may reflect the complex mechanisms by which IL-1 release and responsiveness are controlled in skin.

Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line in vitro.

BACKGROUND: Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications. METHODS: Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines. RESULTS: We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types. CONCLUSION: The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome.

Implementation of enhanced dynamic wedge in the focus rtp system.

The FOCUS RTP system implementation of Varian's enhanced dynamic wedge (EDW) is presented. Calculations of both dose distributions and wedge factors (WFs) are based on segmented treatment tables (STTs). Calculating dose requires a "transmission matrix" derived from an STT to model the modified fluence from the source. The dose calculation is then performed using either the Clarkson or convolution/superposition algorithms. An initial "primary dose/monitor unit (MU) fraction" WF estimate at the weight point of symmetric and asymmetric fields is calculated from the STT as the ratio of MU delivered on the axis of the weight point divided by total MU delivered for the treatment field. In our approach, we go beyond this initial estimate with a "scatter dose" correction. This requires measured 60 degrees WFs for 5 fields. Scatter corrections derived from measured WFs are interpolated for other wedge angles and field sizes in much the same way as arbitrary wedge angle STTs are derived from a "golden STT" using the "ratio of tangents" formalism. Dose comparisons with measured distributions show good agreement to within 3% or 3 mm for 6-MV beams and all EDW angles. Agreement with measurements to within 1% is obtained for WFs in all symmetric and asymmetric fields for 6- and 10-MV beams. For large wedge angles and field sizes, this represents a significant improvement over the 3% to 4% errors often observed using the MU fraction model alone.

beta1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms.

In keratinocytes, the beta1 integrins mediate adhesion to the extracellular matrix and also regulate the initiation of terminal differentiation. To explore the relationship between these functions, we stably infected primary human epidermal keratinocytes and an undifferentiated squamous cell carcinoma line, SCC4, with retroviruses encoding wild-type and mutant chick beta1 integrin subunits. We examined the ability of adhesion-blocking chick beta1-specific antibodies to inhibit suspension-induced terminal differentiation of primary human keratinocytes and the ability of the chick beta1 subunit to promote spontaneous differentiation of SCC4. A D154A point mutant clustered in focal adhesions but was inactive in the differentiation assays, showing that differentiation regulation required a functional ligand-binding domain. The signal transduced by beta1 integrins in normal keratinocytes was "do not differentiate" (transduced by ligand-occupied receptors) as opposed to "do differentiate" (transduced by unoccupied receptors), and the signal depended on the absolute number, rather than on the proportion, of occupied receptors. Single and double point mutations in cyto-2 and -3, the NPXY motifs, prevented focal adhesion targeting without inhibiting differentiation control. However, deletions in the proximal part of the cytoplasmic domain, affecting cyto-1, abolished the differentiation-regulatory ability of the beta1 subunit. We conclude that distinct signaling pathways are involved in beta1 integrin-mediated adhesion and differentiation control in keratinocytes.

Inactivation and regulation of the aerobic C(4)-dicarboxylate transport (dctA) gene of Escherichia coli.

The gene (dctA) encoding the aerobic C(4)-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and the o157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti and Rhizobium leguminosarum H(+)/C(4)-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C(4)-dicarboxylate transport system (DctQ, DctM, and DctP) of Rhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactivated by inserting Sp(r) or Ap(r) cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was approximately 10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C(4)-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C(4)-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive with respect to C(4)-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice.

Previous attempts to achieve long-term gene expression in retrovirally transduced human epidermal keratinocytes in vivo have been largely unsuccessful. This has been variously attributed to a failure to target epidermal stem cells, suboptimal grafting conditions or inactivation of the retroviral vector. In an attempt to overcome these problems we expressed the chick beta 1 integrin subunit in primary human epidermal keratinocytes, which allowed us to monitor retroviral gene expression on a cell-by-cell basis. We describe optimised methods for selecting high-titre amphotropic packaging cells and for infecting keratinocytes in culture. When transduced cells were grafted into mice, graft survival was comparable in nude and SCID mice, but it was essential to combine the keratinocytes with a dermal substrate. Using these methods the majority of keratinocytes expressed the chick beta 1 integrin subunit for at least 16 weeks after grafting. We conclude that epidermal keratinocytes are attractive recipient cells for gene therapy.

The bombesin/GRP receptor transfected into Rat-1 fibroblasts couples to phospholipase C activation, tyrosine phosphorylation of p125FAK and paxillin and cell proliferation.

Bombesin elicits multiple signalling pathways in various cell types. It is not clear, however, whether these responses are mediated by a single receptor subtype or by different subtypes that couple preferentially to specific pathways. To resolve this we transfected the mouse bombesin/GRP receptor into Rat-1 fibroblasts and investigated the pathways activated by bombesin. Expression of the transfected receptors was verified by binding of (125I)GRP and two clones were selected, BOR5 and BOR15. Bombesin stimulation of BOR5 and BOR15 cells caused intracellular Ca2+ mobilisation and increased the phosphorylation of 80K/MARCKS, a prominent protein kinase C substrate. The transfected receptor conferred a proliferative response to bombesin demonstrated by incorporation of (3H) thymidine after 18 h and an increase in total cell numbers after 1-2 days. In BOR5 and BOR15 cells, bombesin rapidly stimulated the tyrosine phosphorylation of multiple proteins Mr 110 000-130 000 and 70 000-80 000 including p125fak and paxillin, at low concentrations (half maximum 0.3 nM). The specific bombesin/GRP receptor antagonist, D-F5-Phe6, D-Ala11-Bombesin (6-13)OMe, inhibited all the above responses. These results show that phospholipase C activation, cell growth and tyrosine phosphorylation emanate from a single class of bombesin receptor.

Mitogenic signaling by transfected neuromedin B receptors in Rat-1 cells.

The rat neuromedin B (NMB) receptor was expressed in Rat-1 fibroblasts to elucidate the signaling pathways and mitogenic effects mediated by this seven-transmembrane domain receptor. Receptor expression was verified by ligand binding and Ca2+ mobilization, which were blocked by the NMB receptor antagonist D-Nal-Cys-Tyr-D-Trp-Orn-Val-Cys-Nal-NH2. NMB acted as a potent growth factor promoting DNA synthesis and cell proliferation in serum-free medium in Rat-1 cells transfected with the NMB receptor. Prior to DNA synthesis, NMB stimulated phosphorylation of 80K/MARCKS, a major substrate of protein kinase C, which could be prevented by the selective protein kinase C inhibitor GF 109203X. Furthermore, NMB induced a rapid p42MAPK activation and tyrosine phosphorylation of multiple proteins including p125FAK and paxillin. The half-maximal concentrations (EC50) of NMB required to induce DNA synthesis (0.7-0.9 nM) and cell proliferation (0.7-1 nM) paralleled the Kd for 125I-[D-Tyr0]NMB binding and the EC50 values for the induction of the early signaling events. Thus, NMB can activate multiple signal transduction pathways and act as a sole mitogen through its receptor expressed in Rat-1 fibroblasts.

The human CCKB/gastrin receptor transfected into rat1 fibroblasts mediates activation of MAP kinase, p74raf-1 kinase, and mitogenesis.

The human cholecystokinin (CCK)B/gastrin receptor was stably transfected into Rat1 fibroblasts to examine the signaling pathways mediated by this seven-transmembrane, G protein-linked receptor. We report here that binding of CCK-8 or gastrin to the CCKB/gastrin receptor induced phosphoinositide breakdown and led to a rapid, transient, and concentration-dependent increase in intracellular Ca2+, which was completely blocked by a specific CCKB receptor antagonist. The peptides also stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Both CCK-8 and gastrin induced a dose- and time-dependent activation of MAP kinase and p74raf-1 kinase in the transfected Rat1 cells. These effects could be dissociated from protein kinase C activation and were not dependent on a functional Gi protein. Finally, both CCK-8 and gastrin induced DNA synthesis in Rat1 cells transfected with the human CCKB/gastrin receptor through a pertussis toxin-insensitive pathway. These results indicate that the neuropeptides gastrin and CCK can activate multiple signal transduction pathways and act as sole mitogens by binding to the CCKB/gastrin receptor transfected into Rat1 fibroblasts.

Overexpression of the myristoylated alanine-rich C-kinase substrate in Rat1 cells increases sensitivity to calmodulin antagonists.

The acidic 80-kDa myristoylated alanine-rich C-kinase substrate protein (80-kDa MARCKS) is the major protein-kinase-C substrate in rodent fibroblasts. To elucidate its function, we transfected the cDNA coding for the 80-kDa MARCKS protein into Rat1 fibroblasts. One clone, called Rat1-80K, expressed 4.5 +/- 0.8-fold and 9.5 +/- 1.5-fold higher levels of 80-kDa MARCKS protein under quiescent and growing conditions, respectively, compared to mock or untransfected control cells. Southern-blot and Northern-blot analyses of Rat1-80K showed intact integration and correct transcription of the introduced 80-kDa MARCKS gene. The overexpressed 80-kDa MARCKS protein was phosphorylated and translocated from the membrane to the cytoplasmic fraction. Since 80-kDa MARCKS has been described as a calmodulin-binding protein in in vitro studies, we investigated the effects of the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and triflouperazine on the entry into the S-phase of the cell cycle in intact cells. DNA synthesis by Rat1-80K cells was more sensitive to either N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or triflouperazine than that of control cells. Our results suggest that overexpression of the 80-kDa MARCKS protein reduces the free concentration of calmodulin in the cell.

Expression of the major protein kinase C substrate, the acidic 80-kilodalton myristoylated alanine-rich C kinase substrate, increases sharply when Swiss 3T3 cells move out of cycle and enter G0.

The expression of the major protein kinase C (PKC) substrate, originally called "80K" for acidic SDS/PAGE-observed 80-kDa PKC substrate and now called "MARCKS" for myristoylated alanine-rich C kinase substrate, in Swiss 3T3 fibroblasts changes strikingly (15- to 22-fold) during transitions of cell growth. Quiescent cells in G0 express high levels of MARCKS mRNA and protein. However, plating these cells in fresh medium at low density to stimulate multiple rounds of cell division caused a striking down-regulation of MARCKS expression. The mRNA level declined to a minimum of 4.5% compared with quiescent control cells 6 hr after plating, and protein levels declined during the same period to 6.5% of the control value. This rapid down-regulation was independent of PKC activation and length of exposure to trypsin (1-10 min) but required plating in medium containing fresh serum. MARCKS mRNA and protein levels remained down-regulated for 3 days, during which time the cells were actively progressing through the cell cycle as judged by fluorescence-activated cell sorting analysis. However, on reaching quiescence, the expression of MARCKS mRNA and protein increased markedly. Furthermore, the rate of recovery of MARCKS mRNA and protein levels was shown to be dependent on the supply of serum-derived growth factors in the medium. Addition of hydroxyurea to arrest the cells in S phase or at the G1/S boundary rather than G0 completely prevented the recovery of MARCKS protein. The down-regulation of MARCKS following plating and its serum-dependent recovery was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The results suggest that MARCKS may play a role in the regulation of entry and exit of cells from G0.

Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS, of protein kinase C.

Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (IK) and the M-current (IM). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E1 (PGE1) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: IK, a small current through a "T-like" Ca2+ channel, and no M-current. Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22 +/- 7%. In contrast to the stimulation of IK observed with cardiac cells, PKC activation produced only a small inhibition of IK, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of IK in neuroblastoma cells.

Immunoprecipitation of viral polypeptides of equid herpesvirus 1 and 4 by serum from experimentally infected ponies.

Sera from two sibling groups of ponies experimentally infected with Equid herpesvirus 1 or 4 (EHV-1 or 4) were used to investigate which viral polypeptides (VPs) of EHV-1 and EHV-4 were recognised. Recognition was detected as early as 8 d.p.i. and thereafter. The polypeptides of EHV-1 (labelled with 35S-methionine) immunoprecipitated (IIP) by sera from both groups had Mr of 148, 138, 123, 117, 110, 77-79, 70, 55, 49-50, 47, 40 and 35-37 kDa respectively. Of these VP148K (VP9 nucleocapsid) gave the maximum precipitation, followed by 117 and 77-79 kDa. The latter were confirmed by monoclonal antibodies as the gB homologue of Herpes-simplex virus (HSV). With EHV-4 the homologous VPs precipitated were similar to those of EHV-1. However, instead of the precipitated VP55K of EHV-1, there were two faint bands of Mr 60 and 55 kDa, neither of significant density. Bands at 123 and 70 kDa were absent. High MW polypeptides (>200 kDa) were not significant and infrequently seen with both viruses. Labelling EHV-1 with 3H glucosamine indicated that viral glycoproteins (VGPs) at an Mr of 79-88 kDa (equivalent to gB and gC) were most commonly recognised in homologous EHV-1 IIP and at 83 kDa (gC) in heterologous IIP. The EHV-4 immunoprecipitated VGPs were at 230-300 kDa with bands at 290 kDa and 250 kDa. Also detected were bands at 100, 123, 79-88, 58-61K and 54-55 kDa. The 79-88 kDa polypeptides gave maximum density and were considered as homologues of HSV gB and gC. Thus the overall profile indicated that following experimental infection the major nucleocapsid protein of 148K, and the gB analogues of 117 and 77 kDa were the most antigenic in experimental infections of ponies with either EHV-1 or 4 and that these showed reciprocal precipitation.