Sedimentation rate and suPAR in relation to disease activity and mortality in patients with tuberculosis.

OBJECTIVE: To investigate how levels of the soluble urokinase plasminogen activator receptor (suPAR) and erythrocyte sedimentation rate (ESR) correlate with disease activity and prognosis in pulmonary tuberculosis (PTB).DESIGN: This was a retrospective analysis of patients with active PTB (n = 500) in Gondar, Ethiopia, for whom the suPAR (n = 301) and ESR (n = 330) were analysed at the start of treatment. Both biomarkers were available for 176 patients. Human immunodeficiency virus (HIV) status, chest X-ray (CXR) findings, classification according to the clinical TBscore and treatment outcome were all recorded.RESULTS: In a multivariable logistic regression analysis adjusted for age, sex and HIV status, surrogate markers of disease activity such as advanced CXR patterns correlated with increased levels of suPAR (adjusted OR [aOR] 8.24, P < 0.001) and of ESR (aOR 1.63, P = 0.030), whereas ESR only correlated significantly with a TBscore >6 points. Increased levels of both suPAR and ESR were associated with unsuccessful treatment outcomes (aOR 2.93, P = 0.013; aOR 2.52, P = 0.025). The highest quartile of suPAR (aOR 13.3, P = 0.029) but not ESR levels correlated independently with increased mortality.CONCLUSION: SuPAR and ESR levels correlate with disease activity in PTB; however, the clinical role of these potentially prognostic biomarkers needs to be verified in prospective studies.

A proteomic study of serum from children with autism showing differential expression of apolipoproteins and complement proteins.

Modern methods that use systematic, quantitative and unbiased approaches are making it possible to discover proteins altered by a disease. To identify proteins that might be differentially expressed in autism, serum proteins from blood were subjected to trypsin digestion followed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) on time-of-flight (TOF) instruments to identify differentially expressed peptides. Children with autism 4-6 years of age (n=69) were compared to typically developing children (n=35) with similar age and gender distributions. A total of 6348 peptide components were quantified. Of these, five peptide components corresponding to four known proteins had an effect size >0.99 with a P<0.05 and a Mascot identification score of 30 or greater for autism compared to controls. The four proteins were: Apolipoprotein (apo) B-100, Complement Factor H Related Protein (FHR1), Complement C1q and Fibronectin 1 (FN1). In addition, apo B-100 and apo A-IV were higher in children with high compared to low functioning autism. Apos are involved in the transport of lipids, cholesterol and vitamin E. The complement system is involved in the lysis and removal of infectious organisms in blood, and may be involved in cellular apoptosis in brain. Despite limitations of the study, including the low fold changes and variable detection rates for the peptide components, the data support possible differences of circulating proteins in autism, and should help stimulate the continued search for causes and treatments of autism by examining peripheral blood.

The molecular basis of CaMKII function in synaptic and behavioural memory.

Long-term potentiation (LTP) in the CA1 region of the hippocampus has been the primary model by which to study the cellular and molecular basis of memory. Calcium/calmodulin-dependent protein kinase II (CaMKII) is necessary for LTP induction, is persistently activated by stimuli that elicit LTP, and can, by itself, enhance the efficacy of synaptic transmission. The analysis of CaMKII autophosphorylation and dephosphorylation indicates that this kinase could serve as a molecular switch that is capable of long-term memory storage. Consistent with such a role, mutations that prevent persistent activation of CaMKII block LTP, experience-dependent plasticity and behavioural memory. These results make CaMKII a leading candidate in the search for the molecular basis of memory.

Massive cavitation of solid pulmonary metastatic lesions in a breast cancer patient: a case report.

A 40-year-old female with metastatic breast cancer developed multiple lung nodules some of them with cavitations. Following treatment with Taxol/Herceptin most of the lesions disappeared and in many cavity lesions appeared. There was no further change in the appearance of lung lesions.

Regulation of signal transduction by protein targeting: the case for CaMKII.

Protein targeting is increasingly being recognized as a mechanism to ensure speed and specificity of intracellular signal transduction in a variety of biological systems. Conceptually, this is of particular importance for second-messenger-regulated protein kinases with a broad spectrum of substrates, such as the serine/threonine protein kinases PKA, PKC, and CaMKII (cyclic-AMP-dependent protein kinase, Ca(2+)-phospholipid-dependent protein kinase, and Ca(2+)/calmodulin-dependent protein kinase II). The activating second messengers of these enzymes can be produced or released in response to a large variety of "upstream" signals, and they can, in turn, regulate a large variety of "downstream" proteins. Targeting, e.g., via anchoring proteins, can link certain incoming stimuli with specific outgoing signals by restricting the subcellular compartment at which activation and/or action of a signaling molecule can take place. Elegant research on PKA and PKC reinforced the biological importance of such mechanisms. We will focus here on CaMKII, as recent advances in the understanding of its targeting have some significant general implications for signal transduction. The interaction of CaMKII with the NMDA receptor, for instance, shows that a targeting protein can not only specify the subcellular localization of a signaling effector, but can also directly influence its regulation.

Kinetic and pharmacological properties of GABA(A) receptors in single thalamic neurons and GABA(A) subunit expression.

Synaptic inhibition in the thalamus plays critical roles in sensory processing and thalamocortical rhythm generation. To determine kinetic, pharmacological, and structural properties of thalamic gamma-aminobutyric acid type A (GABA(A)) receptors, we used patch-clamp techniques and single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in neurons from two principal rat thalamic nuclei-the reticular nucleus (nRt) and the ventrobasal (VB) complex. Single-channel recordings identified GABA(A) channels with densities threefold higher in VB than nRt neurons, and with mean open time fourfold longer for nRt than VB [14.6 +/- 2.5 vs. 3.8 +/- 0.7 (SE) ms, respectively]. GABA(A) receptors in nRt and VB cells were pharmacologically distinct. Zn(2+) (100 microM) reduced GABA(A) channel activity in VB and nRt by 84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to 77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR revealed subunit heterogeneity between nRt and VB cells. VB neurons expressed alpha1-alpha3, alpha5, beta1-3, gamma2-3, and delta, while nRt cells expressed alpha3, alpha5, gamma2-3, and delta. Both cell types expressed more subunits than needed for a single receptor type, suggesting the possibility of GABA(A) receptor heterogeneity within individual thalamic neurons. beta subunits were not detected in nRt cells, which is consistent with very low levels reported in previous in situ hybridization studies but inconsistent with the expected dependence of functional GABA(A) receptors on beta subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can make no conclusion regarding beta subunits, our findings do support alpha subunits, possibly alpha1 versus alpha3, as structural determinants of channel deactivation kinetics and clonazepam sensitivity. As the gamma2 and delta subunits previously implicated in Zn(2+) sensitivity are both expressed in each cell type, the observed differential Zn(2+) actions at VB versus nRt GABA(A) receptors may involve other subunit differences.

Congenital insensitivity to pain with anhidrosis (CIPA): the spectrum of radiological findings.

BACKGROUND: Congenital insensitivity to pain with anhidrosis (CIPA) is an exceedingly rare, hereditary, sensory autonomic neuropathy (HSAN). AIM: To evaluate the various skeletal manifestations and cranial CT features in children affected by CIPA. MATERIALS AND METHODS: In the semidesert area of the Negev, the Bedouin tribes constitute a closed society where consanguineous marriages are the custom. This has resulted in a group of 20 children being affected by this rare autosomal recessive HSAN. The skeletal surveys and CT scans of these 20 Bedouin patients, 12 girls and 8 boys, ages ranging between 1 month and 8 years, were retrospectively analysed. Cranial CT scans were performed in ten children because of neonatal hypotonia and psychomotor retardation. The skeletal findings were classified as follows: fractures, joint deformities, joint dislocations, osteomyelitis, avascular necrosis and acro-osteolysis. RESULTS: All 20 patients had fractures of the extremities and acro-osteolysis of the fingers. Six had joint deformities. Three children had recurrent hip joint dislocations and another three had avascular necrosis. Ten patients presented with osteomyelitis of the limbs, acetabulum and scapula. The cranial CT scans disclosed mild brain volume loss with some ventriculomegaly. CONCLUSIONS: CIPA is a severe autosomal recessive condition that leads to self-mutilation early in life and to fractures, osteomyelitis and limb amputation in older children. Mental retardation is common. Death from hyperpyrexia occurs in almost 20 % of patients in the first 3 years of life.

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.

Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.

Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.

Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II.

Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr(286) results in calmodulin (CaM) trapping, a >10,000-fold decrease in the dissociation rate of CaM from the enzyme. Here we present the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase II. We measured dissociation kinetics of CaM and CaM kinase II proteins by using a fluorescently modified CaM that is sensitive to binding to target proteins. In low [Ca(2+)], the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type. In high [Ca(2+)], the CaM mutations E120A, M124A, and E120A/M124A and the CaM kinase II mutations F293A, F293E, N294A, N294P, and R297E increased dissociation rate constants by factors ranging from 2.3 to 116. We have also identified residues in CaM and CaM kinase II that interact in the trapped state by mutant cycle-based analysis, which suggests that interactions between Phe(293) in the kinase and Glu(120) and Met(124) in CaM specifically stabilize the trapped CaM-CaM kinase II complex. Our studies further show that Phe(293) and Asn(294) in CaM kinase II play dual roles, because they likely destabilize the low affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.

Calcium regulation of exocytosis in PC12 cells.

The calcium (Ca(2+)) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 m sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca(2+) was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca(2+) was present during the priming stage as well as the triggering, suggesting that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca(2+)-triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca(2+) sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca(2+)-binding were not able to enhance release.

Solution and chip arrays in protein profiling.

The industrialization of proteomics demands reproducible, robust and high-throughput profiling technologies that current two-dimensional gel electrophoresis cannot achieve. New technologies in protein arrays, either on chips or with self-encoded elements in solution, hold much promise for interrogating the diverse and immense proteome.

Upper airway obstruction-related sleep apnea in a child with thalassemia intermedia.

Obstructive sleep apnea can be caused by hypertrophy of tonsils and adenoids or neuromuscular diseases. The authors describe a child with thalassemia intermedia in whom severe obstructive sleep apnea syndrome developed. Computed tomography scanning revealed an obstruction of the nasopharynx resulting from extramedullary hematopoiesis. The child was treated with hydroxyurea and blood transfusions. Relief of symptoms was noted 1.5 months after initial treatment. Extramedullary hematopoiesis causes sleep apnea syndrome in thalassemic patients, and the treatment of hydroxyurea and blood transfusion for extramedullary hematopoiesis should be further studied.

Congenital unilateral hydrocephalus--CT findings.

Congenital unilateral hydrocephalus is extremely uncommon with 18 cases previously reported in the English literature. Two additional newborns with unilateral hydrocephalus are presented. The second baby also presented a mega cisterna magna. This unusual association between Dandy-Walker variant and unilateral hydrocephalus has not been previously reported. Following ventriculo-peritoneal shunt, the babies had a normal cognitive neurodevelopment. The role of cranial computed tomography (CT) in diagnosis and follow-up is emphasized.

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and its analogues prevent damage to 2-deoxyribose mediated by ferric iron plus ascorbate.

Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.

Thoracoabdominal peripheral primitive neuroectodermal tumors in childhood: radiological features.

Peripheral primitive neuroectodermal tumors (PNET) are extremely uncommon, malignant neoplasms affecting mostly children and young adults. We retrospectively reviewed the clinical data and radiological studies of four such cases. All cases were pathologically proven. Plain films, US, and CT scans were used. The youngest child had a huge pelvic tumor and two adolescents each had a chest wall (Askin) tumor. The fourth patient had a most unusual location of the PNET in the anterior mediastinum. The CT findings are emphasized. We emphasize that the markedly abnormal CT findings are not specific for PNET.

Inhibition of a mammalian large conductance, calcium-sensitive K+ channel by calmodulin-binding peptides.

The large conductance, calcium-sensitive K+ channel (BKCa channel) is a voltage-activated ion channel in which direct calcium binding shifts gating to more negative cellular membrane potentials. We hypothesized that the calcium-binding domain of BKCa channels may mimic the role played by calmodulin (CaM) in the activation of calcium-CaM-dependent enzymes, in which a tonic inhibitory constraint is removed on CaM binding. To examine such a hypothesis, we used peptides from the autoregulatory domains of CaM kinase II (CK291-317) and cNOS (the constitutive nitric oxide synthase; cNOS725-747) as probes for the calcium-dependent activation of murine BKCa channels transiently expressed in HEK 293 cells. We found that these CaM-binding peptides produced potent, time-dependent inhibition of mammalian BKCa channel current following voltage-dependent activation. Inhibition was observed in both the presence and the absence of cytosolic free calcium. Similar application of CK291-31 had no effect on either the amplitude or kinetics of voltage-dependent, macroscopic currents recorded from rabbit smooth muscle Kv1.5 potassium channels transiently expressed in HEK 293 cells. Cytosolic application of both CK291-317 and tetraethylammonium (TEA) produced an additive and non-competitive block of BKCa current. This finding suggests that the peptide-binding site is distinct (e.g. outside the pore region of the channel) from that of TEA. Our results are thus consistent with a model in which the BKCa channel's voltage-dependent gating process is under an intramolecular constraint that is relieved upon calcium binding. The intrinsic calcium sensor of the channel may thus interact with an inhibitory domain present in the BKCa channel, and by doing so, remove an inhibitory 'constraint' that permits voltage-dependent gating to occur at more negative potentials.

Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions.

Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.

Maggot therapy for the treatment of intractable wounds.

BACKGROUND: Fly maggots have been known for centuries to help debride and heal wounds. Maggot therapy was first introduced in the USA in 1931 and was routinely used there until the mid-1940s in over 300 hospitals. With the advent of antimicrobiols, maggot therapy became rare until the early 1990s, when it was re-introduced in the USA, UK, and Israel. The objective of this study was to assess the efficacy of maggot therapy for the treatment of intractable, chronic wounds and ulcers in long-term hospitalized patients in Israel. METHODS: Twenty-five patients, suffering mostly from chronic leg ulcers and pressure sores in the lower sacral area, were treated in an open study using maggots of the green bottle fly, Phaenicia sericata. The wounds had been present for 1-90 months before maggot therapy was applied. Thirty-five wounds were located on the foot or calf of the patients, one on the thumb, while the pressure sores were on the lower back. Sterile maggots (50-1000) were administered to the wound two to five times weekly and replaced every 1-2 days. Hospitalized patients were treated in five departments of the Hadassah Hospital, two geriatric hospitals, and one outpatient clinic in Jerusalem. The underlying diseases or the causes of the development of wounds were venous stasis (12), paraplegia (5), hemiplegia (2), Birger's disease (1), lymphostasis (1), thalassemia (1), polycythemia (1), dementia (1), and basal cell carcinoma (1). Subjects were examined daily or every second day until complete debridement of the wound was noted. RESULTS: Complete debridement was achieved in 38 wounds (88.4%); in three wounds (7%), the debridement was significant, in one (2.3%) partial, and one wound (2.3%) remained unchanged. In five patients who were referred for amputation of the leg, the extremities was salvaged after maggot therapy. CONCLUSIONS: Maggot therapy is a relatively rapid and effective treatment, particularly in large necrotic wounds requiring debridement and resistant to conventional treatment and conservative surgical intervention.

Structural examination of autoregulation of multifunctional calcium/calmodulin-dependent protein kinase II.

Regulation of Ca(2+)/calmodulin-dependent protein kinase II is likely based on an auto-inhibitory mechanism in which a segment of the kinase occupies the catalytic site in the absence of calmodulin. We analyze potential auto-inhibitory associations by employing charge reversal and hydrophobic-to-charged residue mutagenesis. We identify interacting amino acid pairs by using double mutants to test which modification in the catalytic domain complements a given change in the auto-inhibitory domain. Our studies identify the core pseudosubstrate sequence (residues 297-300) but reveal that distinct sequences centered about the autophosphorylation site at Thr-286 are involved in the critical auto-inhibitory interactions. Individual changes in any of the residues Arg-274, His-282, Arg-283, Lys-291, Arg-297, Phe-293, and Asn-294 in the auto-inhibitory domain or their interacting partners in the catalytic domain produces an enhanced affinity for calmodulin or generates a constitutively active enzyme. A structural model of Ca(2+)/calmodulin-dependent protein kinase II that incorporates these interactions shows that Thr-286 is oriented inwardly into a hydrophobic channel. The model explains why calmodulin must bind to the auto-inhibitory domain in order for Thr-286 in that domain to be phosphorylated and why introduction of phospho-Thr-286 produces the important Ca(2+)-independent state of the enzyme.