Is a wound swab for microbiological analysis supportive in the clinical assessment of infection of a chronic wound?

AIM: To determine whether bacteriological analysis of a wound swab is supportive in the clinical assessment of infection of a chronic wound. METHODS: Patients attending an outpatient wound clinic who had endured a chronic wound for more than 3 weeks were clinically assessed for infection. In addition, standardized wound swabs were taken according to the Levine technique and the microbiological findings of the swabs compared with the clinical assessment of the wounds. RESULTS: There was no significant relationship between the clinical assessments of the chronic wounds and the qualitative or quantitative bacteriological results of the swabs. CONCLUSION: Microbiological analysis of wound swabs taken from chronic wounds to support clinical assessment of the wounds is waste of time and money. It may be preferable to assess chronic wounds clinically, however, validation studies of these signs and symptoms are needed.

Ethical sourcing of dental instruments and materials.

There is evidence that dental instruments and materials are being manufactured in the developing world under poor labour conditions. It is suggested that the level of awareness of the dental team with regard to this is raised and that a culture of greater inquiry into the sourcing of instruments and materials is developed.

Effect of simulated commercial flight on oxygenation in patients with interstitial lung disease and chronic obstructive pulmonary disease.

BACKGROUND: Commercial aircraft cabins provide a hostile environment for patients with underlying respiratory disease. Although there are algorithms and guidelines for predicting in-flight hypoxaemia, these relate to chronic obstructive pulmonary disease (COPD) and data for interstitial lung disease (ILD) are lacking. The purpose of this study was to evaluate the effect of simulated cabin altitude on subjects with ILD at rest and during a limited walking task. METHODS: Fifteen subjects with ILD and 10 subjects with COPD were recruited. All subjects had resting arterial oxygen pressure (PaO2) of >9.3 kPa. Subjects breathed a hypoxic gas mixture containing 15% oxygen with balance nitrogen for 20 minutes at rest followed by a 50 metre walking task. Pulse oximetry (SpO2) was monitored continuously with testing terminated if levels fell below 80%. Arterial blood gas tensions were taken on room air at rest and after the resting and exercise phases of breathing the gas mixture. RESULTS: In both groups there was a statistically significant decrease in arterial oxygen saturation (SaO2) and PaO2 from room air to 15% oxygen at rest and from 15% oxygen at rest to the completion of the walking task. The ILD group differed significantly from the COPD group in resting 15% oxygen SaO2, PaO2, and room air pH. Means for both groups fell below recommended levels at both resting and when walking on 15% oxygen. CONCLUSION: Even in the presence of acceptable arterial blood gas tensions at sea level, subjects with both ILD and COPD fall below recommended levels of oxygenation when cabin altitude is simulated. This is exacerbated by minimal exercise. Resting sea level arterial blood gas tensions are similarly poor in both COPD and ILD for predicting the response to simulated cabin altitude.

Will cancer risk assessment and counseling services survive genetic testing?

With the availability of genetic testing to detect increased hereditary susceptibility to breast and other cancers, Cancer Risk Assessment and Counseling services have come to be viewed by many primarily as a means of obtaining genetic testing and hereditary risk information. The public and healthcare professionals need to be aware that even when genetic testing is not used or is uninformative, families with and without a strong family history of cancer will benefit from Cancer Risk Assessment and Counseling if the process includes assessment of cancer risk, information about cancer etiology, help in dealing with the psychosocial consequences of the cancer experience, and development of emotional and medical coping strategies. Risk assessment services can best serve concerned individuals and their families when sufficient time is allotted for these primary aims, and when genetic testing is seen as one of the useful tools, not the primary goal of such services.

Nitric oxide acts as a postsynaptic signaling molecule in calcium/calmodulin-induced synaptic potentiation in hippocampal CA1 pyramidal neurons.

Postsynaptic injection of Ca(2+)/calmodulin (Ca(2+)/CaM) into hippocampal CA1 pyramidal neurons induces synaptic potentiation, which can occlude tetanus-induced potentiation (Wang and Kelly, 1995). Because Ca(2+)/CaM activates the major forms of nitric oxide synthase (NOS) to produce nitric oxide (NO), NO may play a role during Ca(2+)/CaM-induced potentiation. Here we show that extracellular application of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or postsynaptic co-injection of L-NAME with Ca(2+)/CaM blocked Ca(2+)/CaM-induced synaptic potentiation. Thus, NO is necessary for Ca(2+)/CaM-induced synaptic potentiation. In contrast, extracellular perfusion of membrane-impermeable NO scavengers N-methyl-D-glucamine dithiocarbamate/ferrous sulfate mixture (MGD-Fe) or 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) did not attenuate Ca(2+)/CaM-induced synaptic potentiation, even though MGD-Fe or carboxy-PTIO blocked tetanus-induced synaptic potentiation. This result indicates that NO is not a retrograde messenger in Ca(2+)/CaM-induced synaptic potentiation. However, postsynaptic co-injection of carboxy-PTIO with Ca(2+)/CaM blocked Ca(2+)/CaM-induced potentiation. Postsynaptic injection of carboxy-PTIO alone blocked tetanus-induced synaptic potentiation without affecting basal synaptic transmission. Our results suggest that NO works as a postsynaptic (intracellular) messenger during Ca(2+)/CaM-induced synaptic potentiation.

Attenuation of paired-pulse facilitation associated with synaptic potentiation mediated by postsynaptic mechanisms.

Attenuation of paired-pulse facilitation associated with synaptic potentiation mediated by postsynaptic mechanisms. J. Neurophysiol. 78: 2707-2716, 1997. The relationship between paired-pulse facilitation (PPF) and synaptic potentiation induced by various protocols and their cellular and molecular mechanisms were examined by extracellular field potential and current- or voltage-clamp recordings at CA1 synapses in rat hippocampal slices. Microelectrodes were used for both intracellular recordings and injections of modulators of calcium (Ca2+) and Ca2+/calmodulin (CaM) signaling pathways into postsynaptic neurons. Basal synaptic transmission was not accompanied by changes in PPF. Tetanic stimulation induced long-term potentiation (LTP) of synaptic transmission and attenuated PPF. Experiments stimulating two independent Schaffer collateral/commisural(S/C) pathways showed that PPF attenuation and tetanus-LTP were pathway specific. Postsynaptic injections of pseudosubstrate inhibitors of CaM-dependent protein kinase II and protein kinase C (CaM-KII/PKC), [Ala286]CaMKII286-302 plus PKC19-31, almost completely attenuated tetanus-LTP and reversed PPF attenuation but did not affect synaptic transmission and PPF under basal conditions. Postsynaptic injections of heparin and dantrolene (inhibitors of IP3 and ryanodine receptors at intracellular Ca2+ stores) prevented tetanus-LTP induction and PPF attenuation. Postsynaptic injections of calcineurin (CaN) inhibitors, CaN autoinhibitory peptide (CaN-AIP) or FK-506, enhanced synaptic transmission and decreased PPF. CaN-inhibited synaptic potentiation and PPF attenuation were unaffected by (-)-a-Amino-5-phosphonopentanoic, but blocked by coinjecting 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, heparin plus dantrolene, calmodulin-binding peptide, or [Ala286]CaMKII281-302 plus PKC19-31. PPF attenuation associated with tetanus-LTP or CaN-inhibited synaptic potentiation resulted from smaller increases in the potentiation of the second synaptic responses (R2) compared with the potentiation of the first responses (R1). Our results indicate that PPF attenuation is associated with synaptic potentiation mediated by postsynaptic mechanisms, and postsynaptic Ca2+/CaM signaling pathways play a dual role in synaptic plasticity. CaN activity limits synaptic transmission under basal conditions, whereas the activation of Ca2+-dependent protein kinases enhances synaptic transmission and attenuates PPF at central synapses.

Cellular and molecular bases of memory: synaptic and neuronal plasticity.

Discoveries made during the past decade have greatly improved our understanding of how the nervous system functions. This review article examines the relation between memory and the cellular mechanisms of neuronal and synaptic plasticity in the central nervous system. Evidence indicating that activity-dependent short- and long-term changes in strength of synaptic transmission are important for memory processes is examined. Focus is placed on one model of synaptic plasticity called long-term potentiation, and its similarities with memory processes are illustrated. Recent studies show that the regulation of synaptic strength is bidirectional (e.g., synaptic potentiation or depression). Mechanisms involving intracellular signaling pathways that regulate synaptic strength are described, and the specific roles of calcium, protein kinases, protein phosphatases, and retrograde messengers are emphasized. Evidence suggests that changes in synaptic ultrastructure, dendritic ultrastructure, and neuronal gene expression may also contribute to mechanisms of synaptic plasticity. Also discussed are recent findings about postsynaptic mechanisms that regulate short-term synaptic facilitation and neuronal burst-pattern activity, as well as evidence about the subcellular location (presynaptic or postsynaptic) of mechanisms involved in long-term synaptic plasticity.

Postsynaptic calcineurin activity downregulates synaptic transmission by weakening intracellular Ca2+ signaling mechanisms in hippocampal CA1 neurons.

Protein phosphorylation and dephosphorylation are believed to functionally couple neuronal activity and synaptic plasticity. Our previous results indicated that postsynaptic Ca2+/calmodulin (CaM) signaling pathways play an important role in setting synaptic strength, and calcineurin (CaN) activity limits synaptic responses during basal synaptic transmission and long-term potentiation expression. The inhibition of postsynaptic CaN activity by FK-506 or an autoinhibitory peptide induced synaptic potentiation in hippocampal slices, which occludes tetanus-induced LTP. FK-506-induced synaptic potentiation was expressed in adult but not young rats. To elucidate mechanisms underlying CaN-inhibited synaptic potentiation, we co-injected certain agents affecting Ca2+ signaling pathways with CaN inhibitors into CA1 neurons. Synaptic potentiation induced by FK-506 was significantly attenuated by co-injecting BAPTA, heparin/dantrolene (inhibitors of intracellular Ca2+ release), a CaM-binding peptide, or CaM-KII/PKC pseudosubstrate peptides. These results indicate that postsynaptic CaN activity can downregulate evoked synaptic transmission by weakening intracellular Ca2+ signals and downstream protein kinase activities.

Overexpression of Ca2+/calmodulin-dependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor-induced differentiation.

To examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in cell differentiation and neuronal functions, stable transformants of PC12 cells were established that expressed levels of the alpha-subunit of CaMKII (alpha CaMKII) equivalent to mammalian neurons. The expression of the transfected alpha CaMKII gene or the endogenous beta CaMKII gene was monitored by RNase protection assays, and alpha CaMKII protein expression was determined by Western blots. Several PC12-derived clones expressed amounts of alpha CaMKII mRNA and alpha CaMKII protein similar to that of hippocampal tissues and several orders of magnitude greater than untransfected PC12 cells. CaMKII catalytic activity was four times higher in extracts from alpha CaMKII-overexpressing compared with untransfected PC12 cells. All clones overexpressing alpha CaMKII displayed altered cellular growth and adhesion properties including increased cell-to-substrate adhesion, decreased cell-to-cell adhesion, enhanced contact inhibition, and prolonged survival at confluency. Furthermore, the alpha CaMKII activity in overexpressing PC12 cells inhibited neurite elongation during NGF-induced differentiation. Inhibition of CaMKII activity in vivo with KN-62 caused the morphological phenotypes of alpha CaMKII-overexpressing cells to partially revert to that of untransfected PC12 cells. These results show that alpha CaMKII catalytic activity affects growth, morphology, and NGF-induced differentiation of PC12 cells.

Expired air temperature during prolonged exercise in cool- and hot-humid environments.

The purpose of this investigation was to measure expired air temperature under cool- and hot-humid environmental conditions at rest and during prolonged exercise to: (1) establish if significant increases in body core temperature affected expired air temperature, and (2) to determine if the temperature setting for heating the pneumotachometer in an open-circuit system requires adjustment during prolonged exercise tests to account for changes in expired air temperature. Six male distance runners completed two tests in cool-humid [dry bulb temperature (Tdb) 15.5 (SD 1.3) degrees C, wet bulb temperature (TWb) 12.1 (SD 1.4) degrees C] and hot-humid [Tdb 31.6 (SD 0.6) degrees C, TWb 24.9 (SD 0.6) degrees C, black globe temperature (Tg) 34.3 (SD 0.3) degrees C] environments, running at a velocity corresponding to 65% [67.1 (SD 2.82)%] of their maximal oxygen uptake. Rectal temperature and expired air temperatures were compared at rest, and after 30 min and 60 min of exercise for each environment. The main finding of this investigation was a significant (P < 0.05) but small increase in expired air temperature between the 30-min and 60-min measures in the hot-humid environment. No significant differences in expired air temperature were found between the 30-min and 60-min measures in the cool-humid environment. These findings suggest that: (1) expired air temperature is influenced by elevations in body core temperature during prolonged exercise in hot-humid conditions, and (2) that the temperature setting for heating the head of the pneumotachometer (after determining the appropriate temperature through measuring expired air temperature for the set environmental condition) may require adjustment during prolonged exercise trials in hot-humid environmental conditions.

Effect on the duration of mechanical ventilation of identifying patients capable of breathing spontaneously.

BACKGROUND: Prompt recognition of the reversal of respiratory failure may permit earlier discontinuation of mechanical ventilation, without harm to the patient. METHODS: We conducted a randomized, controlled trial in 300 adult patients receiving mechanical ventilation in medical and coronary intensive care units. In the intervention group, patients underwent daily screening of respiratory function by physicians, respiratory therapists, and nurses to identify those possibly capable of breathing spontaneously; successful tests were followed by two-hour trials of spontaneous breathing in those who met the criteria. Physicians were notified when their patients successfully completed the trials of spontaneous breathing. The control subjects had daily screening but no other interventions. In both groups, all clinical decisions, including the decision to discontinue mechanical ventilation, were made by the attending physicians. RESULTS: Although the 149 patients randomly assigned to the intervention group had more severe disease, they received mechanical ventilation for a median of 4.5 days, as compared with 6 days in the 151 patients in the control group (P=0.003). The median interval between the time a patient met the screening criteria and the discontinuation of mechanical ventilation was one day in the intervention group and three days in the control group (P<0.001). Complications -- removal of the breathing tube by the patient, reintubation, tracheostomy, and mechanical ventilation for more than 21 days -- occurred in 20 percent of the intervention group and 41 percent of the control group (P=0.001). The number of days of intensive care and hospital care was similar in the two groups. Total costs for the intensive care unit were lower in the intervention group (median, $15,740, vs. $20,890 in the controls, P=0.03); hospital costs were lower, though not significantly so (median, $26,229 and $29,048, respectively; P=0.3). CONCLUSIONS: Daily screening of the respiratory function of adults receiving mechanical ventilation, followed by trials of spontaneous breathing in appropriate patients and notification of their physicians when the trials were successful, can reduce the duration of mechanical ventilation and the cost of intensive care and is associated with fewer complications than usual care.

Retinoic acid stimulates alpha-CAMKII gene expression in PC12 cells at a distinct transcription initiation site.

The promoter region of the alpha-subunit of the calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) gene was inserted into a beta-galactosidase (beta-gal) reporter plasmid, and beta-gal activities were examined in neuroblastoma (NB2a) and pheochromocytoma (PC12) cells after transient or stable transfections. The alpha-CaMKII promoter was 12- to 45-fold more active in NB2a compared with PC12 cells after transient or stable transfections. All-trans retinoic acid (RA) stimulated reporter gene expression at both protein and mRNA levels in transfected PC12 cells. RA increased the level of endogenous alpha-CaMKII mRNA in untransfected PC12 cells by 4.4-fold. The transcription initiation site(s) (TIS) of the alpha-CaMKII gene in PC12 cells and rat brain was examined by RNase protection assays (RPA) and reverse transcriptase PCRs. The TIS for the alpha-CaMKII/beta-gal reporter gene in transfected PC12 cells was indistinguishable from the TIS+1 in rat hippocampus. In contrast, the only detectable TIS for the alpha-CaMKII gene in untransfected PC12 cells was located near the ATG translation start codon, 147 nucleotides 3' to TIS+1 in hippocampus. This unusual TIS was also the predominant TIS in rat cerebellum. These results suggest that the alpha-CaMKII promoter may contain sequences that respond directly or indirectly to RA. In addition, the unusual TIS of the alpha-CaMKII gene in PC12 cells and rat cerebellum may contribute to the very low expression of this gene compared with that in hippocampus.

The balance between postsynaptic Ca(2+)-dependent protein kinase and phosphatase activities controlling synaptic strength.

The activities of protein kinases and phosphatases are believed to regulate neuronal activity and synaptic plasticity in brain. Numerous in vivo and in vitro studies have shown that synaptic strength appears stable under basal conditions and during long-term potentiation (LTP) expression. This may reflect a balance between protein kinase and phosphatase activities. To provide experimental evidence for this hypothesis, and based on our knowledge that Ca2+/CaM activates protein kinases and phosphatases and that postsynaptic Ca2+/CaM signal pathways play important roles in synaptic plasticity, we examined the contribution of postsynaptic Ca(2+)-dependent protein kinases and calcineurin (CaN) in regulating synaptic strength. We show that inhibiting postsynaptic Ca2+/CaM-dependent protein kinase II (CaM-KII) and Ca2+/phospholipitidyserine-dependent protein kinase (PKC) in hippocampal CA1 neurons attenuates significantly the expression of LTP, but not basal synaptic transmission. On the other hand, the inhibition of postsynaptic CaN enhances synaptic transmission at potentiated and naive synapses, and increases significantly the magnitude of synaptic potentiation during the induction phase of LTP. These results indicate that postsynaptic CaM-KII and PKC activities are essential for maintaining LTP expression, but CaN activity limits synaptic strength at stable levels during both basal and potentiated synaptic transmission; that is, the dynamic balance between protein phosphorylation and dephosphorylation that sets physiological synaptic strength is dominated by CaN activity.

Regulation of synaptic facilitation by postsynaptic Ca2+/CaM pathways in hippocampal CA1 neurons.

1. Current- and voltage-clamp recordings with simultaneous field potential recordings were used to study the cellular and molecular mechanisms that contribute to synaptic facilitation at CA1 synapses in rat hippocampal slices. Microelectrodes used for intracellular recordings were also used to inject modulators of intracellular signal pathways into postsynaptic CA1 neurons. 2. Paired-pulse stimulation at constant stimulus intensity was used to analyze the relationship between the first evoked response (R1) and the absolute value of paired-pulse synaptic facilitation (R2-R1). The magnitudes of these two measures were inversely correlated. Compared with synapses that control motor functions, the synapses of CA1 pyramidal neurons did not exhibit accumulative synaptic facilitation during repetitive stimulation, which is often believed to be mediated by presynaptic residual Ca2+. 3. During studies on the cellular location of mechanisms contributing to synaptic facilitation, we observed that postsynaptic injections of 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid or [Ala286]CaMKII281-302 [a Ca2+/calmodulin-dependent protein kinase II (CaM-KII) inhibitor peptide] prevented the decreases in paired-pulse facilitation (PPF) and synaptic potentiation induced by elevating extracellular Ca2+. These results show that raising extracellular Ca2+ enhances synaptic transmission in part by activating postsynaptic Ca2+ signal pathways. 4. The injection of Ca2+/calmodulin (CaM) into postsynaptic neurons significantly decreased PPF in 50 of 57 experiments while inducing synaptic potentiation; the Ca2+/CaM-induced synaptic potentiation and PPF attenuation occluded subsequent high Ca(2+)-induced enhancements of synaptic transmission. The changes in PPF induced by postsynaptic injections of Ca2+/CaM were inversely correlated with R1 potentiation. 5. The decreases in PPF induced by postsynaptic Ca2+/CaM injections were prevented by coinjecting pseudosubstrate inhibitors or substrate peptides of CaM-KII and protein kinase C (PKC), and were reversed by subsequent application of cyclothiazide (a blocker of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor desensitization). 6. Our results reveal that postsynaptic Ca2+/CaM signal pathways can modulate synaptic facilitation in the CNS, and the activities of CaM-KII and PKC are involved in this modulation. The physiological significance of such modulation is that synaptic strength could be potentiated by activation of Ca2+/CaM pathways during integration of important sensory input (e.g., learning and memory), whereas decreases in synaptic facilitation may protect synaptic transmission during extreme stimulation so that neuronal signal mechanisms can more accurately code neural information.

Glutamate-induced long-term potentiation enhances spontaneous EPSC amplitude but not frequency.

1. Many examples of long-term potentiation (LPT) are induced by repetitive electrical stimulation of presynaptic axons. LTP also is induced by direct glutamate iontophoresis (1 M, 1-2 microA, 10 s) onto postsynaptic neurons in hippocampal slices without evoked presynaptic stimulation; this form of LTP is called "ionto-LTP". The studies herein test the hypothesis that ionto-LTP is expressed primarily through postsynaptic mechanisms. 2. Whole cell recordings were used to examine the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in CA1 pyramidal neurons. sEPSCs were composed of an equal mixture of tetrodotoxin (TTX)-insensitive miniature EPSCs and EPSCs that appeared to result from spontaneous action potentials (i.e., TTX-sensitive EPSCs). The detection of all sEPSCs was virtually eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM), suggesting that sEPSCs were glutamate-mediated synaptic events. 3. Changes in the amplitude and frequency of sEPSCs were examined during the expression of ionto-LTP to obtain new information about the cellular location of mechanisms involved in synaptic plasticity. Our findings show that ionto-LTP expression results in increased sEPSC amplitude in the absence of lasting increases in sEPSC frequency. 4. Potentiation of sEPSC amplitude without changes in sEPSC frequency has been previously interpreted to be due to postsynaptic mechanisms. Although this interpretation is supported by findings from peripheral synapses, its application to the central nervous system is unclear. We have considered alternative mechanisms. Models based on increased release probability for action potential dependent transmitter release appeared insufficient to explain our results. The most straightforward interpretation of our results is that LTP induced by glutamate iontophoresis on dendrites of CA1 pyramidal neurons is mediated largely by postsynaptic changes.

Postsynaptic injection of CA2+/CaM induces synaptic potentiation requiring CaMKII and PKC activity.

CA2+-regulated protein kinases play critical roles in long-term potentiation (LTP). To understand the role of Ca2+/calmodulin (CaM) signaling pathways in synaptic transmission better, Ca2+/CaM was injected into hippocampal CA1 neurons. Ca2+/CaM induced significant potentiation of excitatory synaptic responses, which was blocked by coinjection of a CaM-binding peptide and was not induced by injections of Ca2+ or CaM alone. Reciprocal experiments demonstrated that Ca2+/CaM-induced synaptic potentiation and tetanus-induced LTP occluded one another. Pseudosubstrate inhibitors or high-affinity substrates of CaMKII or PKC blocked Ca2/CaM-induced potentiation, indicating the requirement of CaMKII and PKC activities in synaptic potentiation. We suggest that postsynaptic levels of free Ca2+/CaM is a rate limiting factor and that functional cross-talk between Ca2+/CaM and PKC pathways occurs during the induction of LTP.

LTP induced by activation of voltage-dependent Ca2+ channels requires protein kinase activity.

We have examined the requirement for protein kinase activity in long-term potentiation (LTP) induced by activation of voltage-dependent Ca2+ channels (VDCCs) in hippocampal slices. We previously demonstrated that LTP induced by application of the K+ channel blocker tetraethylammonium (TEA-LTP) consisted of two distinct components, an NMDA receptor-dependent component and a VDCC-dependent component. The results herein demonstrate that both the NMDA and VDCC-dependent components of TEA-LTP are blocked by K-252a, a broad spectrum protein kinase inhibitor. Furthermore, VDCC-dependent TEA-LTP is attenuated by KN-62, a specific inhibitor of Ca2+/calmodulin dependent protein kinase II (CaM-KII). These results demonstrate that LTP induced by VDCC activation requires protein kinase activity and suggest that different routes of postsynaptic Ca2+ influx activate protein kinases to trigger the induction of LTP but that these enzyme systems may be contained in different cell compartments.

A critical period of protein kinase activity after tetanic stimulation is required for the induction of long-term potentiation.

A critical period of protein kinase activity required for the induction of long-term potentiation (LTP) was determined in area CA1 or hippocampal slices using the broad-range and potent protein kinase inhibitors K-252a and staurosporine. As reported previously, K-252a and staurosporine blocked LTP induction when applied before, during, and after high-frequency stimulation (HFS). In contrast, K-252a did not block LTP when applied only before and during HFS and washed out immediately after HFS. K-252a and staurosporine both attenuated LTP magnitude when applied immediately after or as late as 5 min after HFS. However, K-252a applications beginning 30-45 min after HFS did not affect LTP expression significantly. K-252a had no detectable effect on isolated N-methyl-D-aspartate (NMDA) receptor-mediated EPSPs but significantly inhibited the in situ phosphorylation of specific hippocampal proteins (synapsin I, MARCKS, and B-50). In addition, K-252a attenuated 4 beta-phorbol-12,13-dibutyrate (PDBu)-enhanced synaptic transmission. Our results indicate that there is a critical period of protein kinase activity required for LTP induction that extends for approximately 20 min after HFS. In addition, our results suggest that protein kinase activity during and immediately after HFS is not sufficient for LTP induction. These results provide new information about the mechanisms that underlie LTP induction and expression and provide evidence for persistent and/or Ca(2+)-independent protein kinase activity involvement in LTP.

Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element.