A dynamic study of the intensity of mitogenesis in an osteoblast culture in the presence of ultrasonically dispersed hydroxyapatite

Ceramic hydroxyapatite is frequently used in clinical practice nowadays; it is applied in surgical interventions on facial skull bones. This biological material is used as bone substitute and bone connector, but many authors claim that it is devoid of osteoinductor properties. Research is in progress on the effects of a "cold", that is, biochemically active form of ceramic hydroxyapatite on proliferative activity of a number of cell populations. No mitogenesis stimulation was observed during incubation of hydroxyapatite with osteoblast culture; on the contrary, a reduction of proliferative processes intensity was observed. Remembering that hydroxyapatite is a surfactant we repeated this experiment under similar conditions but used a hydroxyapatite molbfication with specific activity increased by two orders in comparison with its close analogs. Mitogenesis intensity was assessed by radiometric method from 3H-thymidine incorporation. The data indicated stimulation of proliferative processes in osteoblast culture.     

Altered impulse activity modifies synaptic physiology and mitochondria in crayfish phasic motor neurons

1. Crayfish phasic motor synapses produce large initial excitatory postsynaptic potentials (EPSPs) that fatigue rapidly during high-frequency stimulation. Periodic in vivo stimulation of an identified phasic abdominal extensor motor neuron (axon 3) induced long-term adaptation (LTA) of neuromuscular transmission: initial EPSP amplitude became smaller and synaptic depression was significantly reduced. We tested the hypothesis that activity-induced synaptic fatigue-resistance seen during LTA was dependent upon, or correlated with, mitochondrial oxidative competence. 2. Periodic unilateral conditioning stimulation of axon 3 entering each of two adjacent homologous abdominal segments (segments 2 and 3) increased the synaptic stamina in both "conditioned" axons; mean final EPSP amplitudes, recorded after 20 min of 5-Hz test stimulation, were significantly larger than those measured with the same protocol from contralateral unstimulated axons. 3. During 5-Hz test stimulation of the conditioned axon 3 of segment 3, acute superfusion with 0.8 mM dinitrophenol or 20 mM sodium azide [inhibitors of oxidative adenosinetriphosphate (ATP) synthesis] produced increased synaptic depression. Drug-free saline superfusion of the conditioned axon 3 of segment 2 in these same animals did not affect the increased synaptic fatigue resistance seen in this segment. Thus both successful induction (in axon 3 of saline-perfused segment 2) and attenuation (in axon 3 of drug-perfused segment 3) of the increased synaptic stamina can be demonstrated with this twin-segment conditioning protocol. 4. Confocal microscopic imaging of mitochondrial rhodamine-123 (Rh123) fluorescence was used to assess relative oxidative competence of conditioned and unconditioned phasic axons. Conditioned phasic axons showed significantly higher mean mitochondrial Rh123 fluorescence than contralateral unstimulated axons. In the same preparations that showed increased postconditioning Rh123 fluorescence, the synaptic fatigue resistance measured from conditioned axon 3 was also significantly greater than that recorded from contralateral unstimulated axon 3. 5. Axotomy of the phasic extensor nerve root (containing axon 3), before in vivo conditioning stimulation of its decentralized segment, prevented induction of both the increased synaptic stamina in axon 3 and the enhanced mitochondrial fluorescence in decentralized motor axons of the nerve root. Hence, induction of both changes requires axonal transport of materials between the soma and the motor synapses of axon 3. 5. Axotomy of the phasic extensor nerve root (containing axon 3), before in vivo conditioning stimulation of its decentralized segment, Prevented induction of both the increased synaptic stamina in axon 3 and the enhanced mitochondrial fluorescence in decentralized motor axons of the nerve root Hence, induction of both changes requires axonal transport of materials between the soma and the motor synapses of axon 3 6. Because mitochondrial Rh123 fluorescence is primarily dependent upon the oxidative activity of these organelles, our findings suggest that conditioning stimulation of phasic extensor axon 3 increases its mitochondrial oxidative competence and that the enhanced synaptic stamina seen during LTA in axon 3 is correlated with, and dependent upon, oxidative activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sol-gel-derived thick-film amperometric immunosensors

Sol-gel processing is used for the first time for the preparation of electrochemical immunosensors. One-step sensor fabrication, based on the coupling of sol-gel and screen-printing technologies, is employed. A low-temperature cured ink is prepared by dispersion of rabbit immunoglobulin G (RIgG), graphite powder, and a binder in the sol-gel solution. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen, which retains its binding properties, and the association reaction is easily detected at the dispersed graphite surface. Use of anti-RIgG labeled with alkaline phosphatase, naphthyl phosphate as the substrate, and amperometric detection at +400 mV (vs Ag/AgCl) results in a low detection limit of 5 ng/mL (32 pM) for the solution antigen. Tailoring the porosity of the ceramic-carbon matrix can be used for tuning the assay performance. The high sensitivity, low cost, durability, and simplicity of the new single-use immunosensors make them well suited for various on-site applications.     

Functional analysis of ZNF85 KRAB zinc finger protein, a member of the highly homologous ZNF91 family

We previously identified the ZNF85 (HPF4) KRAB zinc finger gene, a member of the human ZNF91 family. Here, we show that the ZNF85 gene is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line. Immunocytochemical localization of a panel of beta-Gal/ZNF85 fusion proteins revealed that ZNF85 contains at least one nuclear localization signal located in the spacer region connecting the KRAB domain with the zinc finger repeats. Bacterially expressed ZNF85 zinc finger domain bound strongly and exclusively to DNA in vitro in a zinc-dependent manner. The KRAB(A) domain of the ZNF85 protein and of several other members of the ZNF91 family exhibited repressing activity when tested in Gal4 fusion protein assays. The repression was significantly enhanced by the addition of the KRAB (B) domain, whereas further addition of other conserved regions had no effect. The ZNF85 KRAB(A) and (B) domains in vitro bound several nuclear proteins that might constitute critical cofactors for repression.     

sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast

In the fission yeast Schizosaccharomyces pombe, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein. Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast. In this paper we describe one such component, encoded by the sud1(+) gene. sud1(+) shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast. Cells lacking sud1(+) accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators. Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Deltasud1 cells, but is not the cause of the defect in Cdc18 proteolysis. Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo. Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1. Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles.     

The primary structure of rabbit serum amyloid A protein isolated from acute phase serum

Serum amyloid A (SAA) protein, a sensitive acute phase protein and the precursor of protein AA in secondary amyloid, was purified from pooled acute phase rabbit serum using two different methods: isolation of protein SAA directly by octyl-Sepharose chromatography of total serum, and dissociation and isolation of apoSAA from acute phase high density lipoprotein (HDL). The protein SAA fraction obtained was further purified using gel filtration and ion exchange chromatography. Rabbit protein SAA has 104 amino acid residues, like human SAA, and has a partially blocked N terminus. The highly conserved region from position 33 to position 63 found in SAA from all species studied was confirmed also in rabbit SAA. No microheterogeneities were observed. The amino acid sequence showed extensive N-terminal homology with the rabbit amyloid A protein, except for the microheterogeneity in position 12 in protein AA. It also showed identical amino acid sequence with that deduced from the rabbit cDNA clone pSAA 55. Complete homologies were found with clone SAA 2, except for positions 22 and 78, clone SA8-1, except for positions 22 and 79 and clone SA7-3, except for position 22. This pSAA 55/SA7-3/SA8-1/SAA2-like protein was the only SAA isotype found both in total serum and in the HDL fraction. Isotypes corresponding to other SAA-like genes could not be found in this pool of acute phase rabbit sera.     

Exercise training for intermittent claudication: does it adversely affect biochemical markers of the exercise-induced inflammatory response?

OBJECTIVES: To identify a stable biochemical marker of disease severity in patients with intermittent claudication and to use these findings to assess the effect of therapeutic exercise training. DESIGN: Case-control study: prospective randomised-controlled trial of exercise training. MATERIALS AND METHODS: Plasma fibrinogen, serum amyloid A protein (SAA), C-reactive protein (CRP) and urinary albumin-creatinine ratio (ACR) were measured in 67 claudicants and 15 controls. Twenty-two patients were randomised to supervised exercise training and 17 randomised to observation. Subjects were reviewed at 3, 6 and 12 months. RESULTS: The median (interquartile range) baseline fibrinogen was 3.7 g/l (3.3-4.25) in claudicants and 3.5 g/l (2.9-3.95) in controls (p = 0.045); CRP was 4.7 mg/l (2.2-9.0) and 2.1 mg/l (1.0-2.8), respectively (p < 0.0001); SAA was 72 mg/l (35-132) and 30 mg/l (20-89) (p = 0.0009). Claudicants showed an increased urinary ACR following treadmill exercise (Wilcoxon, p < 0.0001) with no change in controls. Exercise training reduced SAA at 6 months, CRP at 3 months and progressively attenuated the post-exercise increase in ACR. No similar changes were found in controls. CONCLUSIONS: Repetitive low-grade inflammatory events in claudicants lead to elevation of serum acute-phase proteins. Exercise training is associated with symptomatic improvement and reduction inflammatory markers. The concern that exercise has adverse systemic effects therefore seems to be unjustified.     

Social senses: G-protein-coupled receptor signaling pathways in Dictyostelium discoideum

Incubation of phenol-induced cells of the yeast Candida maltosa SBUG 700 with mono- and dichlorophenols resulted in the formation of metabolites of the substrates and of further metabolites not related to the degradation pathway of the substrates. These additional compounds, identified as 4-hydroxyphenylacetic acid (4-HPA), phenylacetic acid (PA), indolylacetic acid (IA) and indolylethanol (i.e.) by means of HPLC and GC/MS, were not excreted in incubation experiments with glucose. The excretion of these metabolites of aromatic amino acid metabolism is not caused by toxic effects of the phenol derivatives, but seems to be a result of carbon and nitrogen starvation of yeast cells.