[Diaphyseal osteoporotic nonunion of the humerus in elderly patients. Surgical treatment and long-term results]. / No-consolidación diafisaria de húmero en pacientes adultos con osteoporosis. Tratamiento quirúrgico y resultados a largo plazo.

INTRODUCTION: to evaluate the long term radiographic and functional results achieved in adult patients with osteoporotic, atrophic, non-unions of the diaphyseal humerus, treated surgically by open reduction and internal fixation with plates and bone graft. MATERIAL AND METHODS: we retrospectively evaluated 22 patients. Patient's age averaged 72 years. Time from initial trauma to definitive surgery averaged 18 months. Eleven patients were smokers, and four had active infection. Pre-operative Constant score and DASH score averaged 23.13 and 81.04, respectively. Pre-operative pain scale averaged 7.45 points. RESULTS: follow-up averaged 69 months. Union was achieved in all cases after an average of 4.68 months. DASH score at last follow-up averaged 20.27 points and Constant score 79.31 points. Analog pain scale averaged 0.77 points. Stabilization was performed using locking blade plates in 12 non-unions, locking compression plates in six cases, and double plating in four non-unions. Patients with active infection were treated in two stages using Masquelet's technique. Bone graft was associated in all cases (cancellous iliac crest autograft in 17, allograft in three, and combined structural allograft and cancellous autograft in two). Two grams of vancomycin powder were associated to the bone graft in all cases. CONCLUSION: the use of open reduction and internal fixation with plates associated to bone graft with local antibiotics, aloud achieving bony union and good predictable long-term objective and subjective functional results in all cases, without major complications or the need of further surgical intervention.

INTRODUCCIÓN: evaluar los resultados radiográficos y funcionales obtenidos a largo plazo en pacientes adultos que presentaron no-consolidaciones atróficas diafisarias de húmero asociadas a osteoporosis; tratadas quirúrgicamente mediante reducción abierta y fijación interna con placas e injerto óseo. MATERIAL Y MÉTODOS: evaluamos retrospectivamente 22 pacientes, con edad promedio de 72 años, el tiempo desde el trauma inicial hasta la cirugía definitiva promedió, 18 meses. Once pacientes eran fumadores y cuatro presentaban infección activa. El score de Constant y el DASH preoperatorios promediaron 23.13 y 81.04, respectivamente. El valor de la escala analógica del dolor preoperatorio promedió 7.45 puntos. RESULTADOS: el seguimiento promedió 69 meses. Se obtuvo la consolidación en todos los casos, luego de un promedio de 4.68 meses. Al último seguimiento, los valores del DASH promediaron 20.27 puntos y el score de Constant promedió 79.31 puntos. La escala analógica del dolor promedió 0.77 puntos. La estabilización se realizó utilizando clavos placa bloqueados en 12 no-consolidaciones, placas bloqueadas de compresión en seis y doble placa en cuatro. Los pacientes con infección activa fueron tratados en dos etapas utilizando la técnica descripta por Masquelet. Se asoció injerto óseo en todas las reconstrucciones (autoinjerto esponjoso de cresta ilíaca en 17, aloinjerto en tres y se combinó aloinjerto estructural con autoinjerto esponjoso en dos). Dos gramos de vancomicina en polvo fueron asociados localmente al injerto óseo. CONCLUSIÓN: la combinación de reducción abierta y fijación interna con placas e injerto óseo permitió obtener la consolidación y resultados funcionales objetivos y subjetivos buenos y predecibles a largo plazo en todos los casos, sin complicaciones mayores ni la necesidad de intervenciones quirúrgicas sucesivas.

Características y resultados de pacientes COVID-19 tratados con ventilación mecánica invasiva en Hospital San Juan de Dios. Marzo-junio 2020

Background: The overall mortality of patients with COVID-19 admitted to intensive care units is approximately 40%. Aim: To describe the characteristics of a cohort of patients with COVID-19 who required invasive mechanical ventilation due to severe hypoxemic acute respiratory failure at a general hospital in Santiago, Chile. Material and Methods: Review of medical records and follow up for 28 days of patients with COVID-19 confirmed by polymerase chain reaction who required invasive mechanical ventilation and who were admitted to the intensive care unit from March 24 to June 7, 2020. Results: Data from 152 patients aged 58 (interquartile range (IQR) 47-65 years (66% men) was analyzed. As of July 5, 36 (24%) had died, 75 (49%) were discharged, 10 (7%) were still on invasive mechanical ventilation, 11 (7%) remained with tracheostomy but without invasive mechanical ventilation, and 20 (13%) were hospitalized in a basic unit. The median time on invasive mechanical ventilation among extubated patients was 14 days (IQR 10-21) and 121 (80%) were in the prone position. Patients who died were older, had a higher frequency of diabetes mellitus and a higher driving pressure at 7 days than those discharged alive from the intensive care unit. Conclusions: In this study mortality was lower than that reported in the first international studies, probably due to the selection of younger patients and greater knowledge of the disease.

[Features and evolution of patients with COVID-19 who required mechanical ventilation]. / Características y resultados de pacientes COVID-19 tratados con ventilación mecánica invasiva en Hospital San Juan de Dios. Marzo-junio 2020.

BACKGROUND: The overall mortality of patients with COVID-19 admitted to intensive care units is approximately 40%. AIM: To describe the characteristics of a cohort of patients with COVID-19 who required invasive mechanical ventilation due to severe hypoxemic acute respiratory failure at a general hospital in Santiago, Chile. MATERIAL AND METHODS: Review of medical records and follow up for 28 days of patients with COVID-19 confirmed by polymerase chain reaction who required invasive mechanical ventilation and who were admitted to the intensive care unit from March 24 to June 7, 2020. RESULTS: Data from 152 patients aged 58 (interquartile range (IQR) 47-65 years (66% men) was analyzed. As of July 5, 36 (24%) had died, 75 (49%) were discharged, 10 (7%) were still on invasive mechanical ventilation, 11 (7%) remained with tracheostomy but without invasive mechanical ventilation, and 20 (13%) were hospitalized in a basic unit. The median time on invasive mechanical ventilation among extubated patients was 14 days (IQR 10-21) and 121 (80%) were in the prone position. Patients who died were older, had a higher frequency of diabetes mellitus and a higher driving pressure at 7 days than those discharged alive from the intensive care unit. CONCLUSIONS: In this study mortality was lower than that reported in the first international studies, probably due to the selection of younger patients and greater knowledge of the disease.

Minimally invasive plate osteosynthesis in distal radius fractures with metaphyseal extension: A series of 13 cases.

UNLABELLED: To evaluate objective and subjective outcomes after minimally invasive volar locked plate fixation of distal radius fractures with metaphyseal extension, we retrospectively evaluated 13 patients with unstable distal radius fractures with metaphyseal extension, treated by minimally invasive volar locked plating. Patients' average age was 41 years. Two volar incisions, 2 to 3cm long, were made; indirect reduction was performed and a volar locked T-plate was placed submuscularly under fluoroscopy guidance. Twelve fractures healed after an average of 2.46 months; one patient needed revision due to a new injury. The plate had to be removed in one patient. On X-rays, radial height averaged 12.78mm, radial inclination averaged 21.34° and volar tilt averaged 8.22°. Flexion averaged 75°, extension 71.5°, pronation 82.08° and supination 83.08°. Grip strength averaged 83.75% of the contralateral wrist. The DASH score averaged 13.91 points and pain assessed on VAS averaged 0.92 points. In unstable distal radius fractures with metaphyseal extension, minimally invasive plate osteosynthesis using volar locked plates led to good reduction and stable fixation, with low pain levels, and good functional and esthetic results. Indirect reduction techniques, fluoroscopy, and restoration of radial length, rotation and alignment, are necessary to achieve these outcomes. LEVEL OF EVIDENCE: IV.

Extensor carpi ulnaris problems at the wrist--classification, surgical treatment and results.

Twenty-eight extensor carpi ulnaris lesions at the wrist were treated surgically between 1990 and 2002. Fifteen patients had an isolated extensor carpi ulnaris tenosynovitis or tendinopathy, five had extensor carpi ulnaris dislocation, four had an extensor carpi ulnaris subluxation and four had an extensor carpi ulnaris rupture. Seventeen patients first developed their symptoms while playing sports. At a mean follow-up of 23 months, twenty-two patients had returned to their previous activities. Seven of the 27 patients had lost more than 30% of their grip strength and five had restricted wrist motion. Two needed an extensor carpi ulnaris tenolysis. Pure isolated extensor carpi ulnaris lesions are rare and associated ulnar sided lesions (eleven triangular fibrocartilage complex tears and four lunotriquetral ligament tears), as well as possible predisposing factors (seven anomalous tendon slips, four ulnar styloid non-unions and one flat extensor carpi ulnaris tendon groove), were frequent. A classification of extensor carpi ulnaris tendon and subsheath lesions was developed to allow the surgeon to adequately evaluate the different components of these lesions.

Intercondylar distal humerus fractures--surgical treatment and results.

INTRODUCTION: Controversy persists concerning the preferred treatment for intercondylar distal humerus fractures. The present study was undertaken to evaluate the clinical results after the surgical treatment of 40 intercondylar distal humerus fractures with an average follow-up of 3.9 years. METHODS: The fractures were classified following the AO/ASIF comprehensive classification. Eight patients presented multiplane fractures. Skeletal traction was used temporarily in two cases. The stabilization method was selected according to the fracture pattern, bone quality and associated lesions. Bone graft was used in seven cases. Fibrin-glue was used in two cases. Unilateral hinged external fixators were used in addition in four cases. Functional assessment was done according to the scoring system of the Orthopedic Trauma Association and additional parameters taken from the system of Jupiter. RESULTS: Final global results were excellent in 13 patients, good in 21, fair in four and poor in two. Complications included three non-unions, two heterotopic ossifications, two internal fixation failures and two lateral condyle resorptions (avascular necrosis). DISCUSSION: Final results are related to the severity of the initial trauma, time elapsed between the accident and definitive surgery, associated lesions, bone quality, precise reconstruction of a smooth and congruent joint surface, surgical technique, implants used, stability obtained and patient cooperation. The type, number and location of the osteosynthesis material must be selected according to the fracture pattern, bone quality and associated lesions.

Resonancia magnética en el estudio de cardiopatías congénitas: rol en la evaluación de las estructuras vasculares extracardiacas / Magnetic resonance in the study of congenital cardiopathies: role in the evaluation of the extracardiac vascular structures

En la actualidad la resonancia magnética ha adquirido cada vez más importancia en el diagnóstico y seguimiento de las cardiopatías congénitas. Entrega información anatómica y más recientemente información funcional. Se presenta la experiencia acumulada entre enero y agosto del año 2002, en cinco casos pediátricos en que la RM cardiaca fue utilizado como método complementario a la ecocardiografía y angiografía. En todos los casos aportó información anatómica precisa, en especial de la anatomía vascular extracardiaca y fue determinante en la conducta terapéutica. Se discute además las características de método e indicaciones.

The genomic structure of two protein kinase CK2alpha genes of Xenopus laevis and features of the putative promoter region.

Protein kinase CK2 is an enzyme that is ubiquitous in eukaryotes. This enzyme, composed of catalytic (alpha and alpha') and regulatory (beta) subunits, is responsible for the phosphorylation of a large number of proteins and is implicated in cell division. Genomic clones coding for the CK2alpha subunit of Xenopus laevis have been isolated. Initial restriction enzyme profiles and subsequent PCR analysis and DNA sequencing indicated that these genomic clones correspond to two different genes. The two genes are highly homologous in the regions of the coding sequence (only 3 amino acid differences) but differ considerable in their intron sequences and lengths. Gene 1 corresponds to the cDNA of XlCK2alpha which had been previously isolated and described. The genomic clone for this gene was truncated. Gene 2 contains the entire coding region for CK2alpha subunit as well as a fragment of 6.4 kb of the 5' upstream region. The exon/intron boundaries of both genes obey the GT/AG rule with the exception of intron V where the less common GC/AG is seen. Comparison of the size of ten coding exons and sites where these are interrupted by introns shows strong conservation with respect to the human CK2alpha gene. RT-PCR analysis of mRNAs from X. laevis ovary, oocytes and early embryos using a specific primer for gene 2 demonstrated that this gene is expressed in these tissues and cells. Analysis of transcription start sites using 5'RACE and RNA from stage VI oocytes demonstrated that there are multiple start sites in the XlCK2alpha mRNA. It was also seen that a noncoding exon 1 is present 4 kb upstream of the translation start site and that alternate splicing occurs in gene 2 to give exon 1 of different lengths. Sequencing of the entire upstream genomic region of gene 2 revealed that there are regions of homology to the sequence of exon 1 of the human CK2alpha gene. Other sequences with consensus to transcription factor binding sites that are seen in the promoter region of human CK2alpha are also found in the X. laevis CK2alpha gene 2. These sites include Ets1, E2F, CCAAT and GC rich regions. No canonical TATA motif is observed.

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2.

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic (alpha and alpha') and regulatory (beta) subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of 'free' CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemagglutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with alpha and beta subunits of Xenopus CK2, with the alpha' subunit of D. rerio, and with Xl CK2alphaA156, which although inactive can bind tightly to CK2beta, and with Xl CK2alphaE75E76, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10-20% of treated cells. Expression of CK2alpha or CK2alphaE75E76 in COS-7 cells caused an increase of 5-7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2beta, the activity increased further to 15-20-fold of the controls. Transfection of CK2beta alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2alphaA156 yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, co-transfection of CK2alpha or CK2alphaE75E76 with CK2alphaA156 caused a 60-70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2alpha subunits. These results can be interpreted as meaning that CK2alphaA156 is a dominant negative mutant that can compete with the other catalytic subunits for the CK2beta subunit. Addition of recombinant CK2beta to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2beta subunit is limiting in the extracts and that an excess of free CK2alpha has been produced in the transfected cells. Transfection of cells with CK2alphaE75E76 results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2alphaE75E76 to heparin increases considerably when it forms part of the holoenzyme CK2alpha2beta2.

Involvement of asparagine 118 in the nucleotide specificity of the catalytic subunit of protein kinase CK2.

Protein kinase CK2 is a heteromeric enzyme with catalytic (alpha) and regulatory (beta) subunits which form an alpha2beta2 holoenzyme and utilizes both ATP and GTP as nucleotide substrate. Site-directed mutagenesis of CK2alpha subunit was used to study this capacity to use GTP. Deletion of asparagine 118 (alpha(deltaN118)) or the mutant alphaN118E gives a 5-6-fold increase in apparent Km for GTP with little effect on the affinity for ATP. Mutants alphaN118A and alphaD120N did not alter significantly the Km for either nucleotide. CK2alphaN118 has an apparent Ki for inosine 5' triphosphate 5-fold higher than wild-type and is very heat labile. These studies complement recent crystallographic data indicating a role for CK2alpha asparagine 118 in binding the guanine base.

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling.

The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.

Interactions of protein kinase CK2 subunits.

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2alpha that has Asp 156 mutated to Ala (CK2alphaA156) is able to bind the CK2beta subunit and to compete effectively in this binding with wild-type subunits alpha and alpha'. The interaction between CK2alphaA156 and CK2beta was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2alphaA156 coprecipitated the beta subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2alphaA156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2alphaA156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2alphaA156 affects the endogenous activity of CK2. Transfection experiments with CK2alpha and beta subunits and CK2alphaA156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2beta indicated that CK2beta is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.

Optimal sequences for non-phosphate-directed phosphorylation by protein kinase CK1 (casein kinase-1)--a re-evaluation.

A variety of synthetic peptides derived from either the inhibitor-2 (I-2) phosphoacceptor sites or the optimal sequences selected in an oriented peptide library have been compared for their susceptibility to phosphorylation by protein kinase CK1 (also termed casein kinase-1). The I-2-derived peptides are by far preferred over the library peptides by both rat liver CK1 (and by the alpha/beta, gamma and delta/epsilon isoforms immunoprecipitated from it) and recombinant Xenopus laevis CK1 alpha. The superiority of the I-2-derived peptides over the library ones is reflected by Vmax values one to two orders of magnitude higher while the Km values are comparable. Individual substitutions of any of the aspartic acids with alanine in the I-2-derived peptide RRKHAAIGDDDDAYSITA is detrimental, producing both a fall in Vmax and an increase in Km which are more pronounced at position n -3, but also quite significant at positions n -4, n -5 and, to a lesser extent, n -6. The unfavourable effect of these substitutions is more evident with rat liver CK1 than with recombinant Xenopus laevis CK1 alpha. The chimeric peptide IGDDDDAY-S-IIIFFA, resulting from the combination of the N-terminal acidic sequence of the I-2 (Ser86) site and the C-terminal hydrophobic cluster selected in the library peptides (MAEFDTG-S-IIIFFAKKK and MAYYDAA-S-IIIFFAKKK) is phosphorylated as efficiently as the I-2-derived peptide in terms of both Km and Vmax. These combined data strongly support the conclusion that, at variance with the optimal sequences selected in the library, optimal non-phosphate-directed phosphorylation of peptide substrates by CK1 critically relies on the presence of a cluster of acidic residues (preferably aspartic acid) upstream from position n -2, while the highly hydrophobic region downstream from serine selected in the library appears to be dispensable. The reason for these discrepancies remains unclear. The possibility that the library data are biased by the invariant elements forming its scaffold (MA-x-x-x-x-x-SI-x-x-x-x-AKKK) would be consistent with the observation that the library-selected peptides, despite their low Km values, fail to compete against the phosphorylation of protein and peptide substrates by CK1, suggesting that they bind to elements partially distinct from those responsible for substrate recognition.

Promiscuous subunit interactions: a possible mechanism for the regulation of protein kinase CK2.

Protein kinase CK2 is a ubiquitous eukaryotic ser/thr protein kinase. The active holoenzyme is a heterotetrameric protein composed of catalytic (alpha and alpha') and regulatory (beta) subunits that phosphorylates many different protein substrates and appears to be involved in the regulation of cell division. Despite important structural studies, the intimate details of the interactions of the alpha catalytic subunits with the beta regulatory subunits are unknown. Recent evidence that indicates that both CK2 subunits can interact promiscuously with other proteins in a manner that excludes the binding of their complementary CK2 partners has opened the possibility that the phosphorylating activity of this enzyme may be regulated in a novel way. These alternative interactions could limit the in vivo availability of CK2 subunits to generate fully active holoenzyme CK2 tetramers. Likewise, variations in the ratio of alpha- and beta-subunits could determine the activity of several phosphorylating and dephosphorylating activities. The promiscuity of the CK2 subunits can be extrapolated to a more widespread phenomenon in which "wild-card" proteins could act as general switches by interacting and regulating several catalytic activities.

Promiscuous subunit interactions: A possible mechanism for the regulation of protein kinase CK2.

Protein kinase CK2 is a ubiquitous eukaryotic ser/thr protein kinase. The active holoenzyme is a heterotetrameric protein composed of catalytic (α and α') and regulatory (ß) subunits that phosphorylates many different protein substrates and appears to be involved in the regulation of cell division. Despite important structural studies, the intimate details of the interactions of the α catalytic subunits with the ß regulatory subunits are unknown. Recent evidence that indicates that both CK2 subunits can interact promiscuously with other proteins in a manner that excludes the binding of their complementary CK2 partners has opened the possibility that the phosphorylating activity of this enzyme may be regulated in a novel way. These alternative interactions could limit the in vivo availability of CK2 subunits to generate fully active holoenzyme CK2 tetramers. Likewise, variations in the ratio of α- and ß-subunits could determine the activity of several phosphorylating and dephosphorylating activities. The promiscuity of the CK2 subunits can be extrapolated to a more widespread phenomenon in which "wild-card" proteins could act as general switches by interacting and regulating several catalytic activities. J. Cell. Biochem. Suppls. 30/31:129-136, 1998. © 1998 Wiley-Liss, Inc.

An inactive mutant of the alpha subunit of protein kinase CK2 that traps the regulatory CK2beta subunit.

Protein kinase CK2 (casein kinase 2) is a ubiquitous Ser/Thr protein kinase involved in cell proliferation. Mutation of the alpha subunit of the Xenopus laevis CK2 to change aspartic acid 156 to alanine (CK2alphaA156) resulted in an inactive enzyme. The CK2alphaA156 mutant, however, binds the regulatory subunit as measured by retention of beta on a nickel chelating column mediated by (His)6-tagged CK2alphaA156. Addition of CK2alphaA156 also caused beta to shift sedimentation in a sucrose gradient from a beta2 dimer (52 kDa) to an alpha2beta2 tetramer (130,000 kDa). CK2alphaA156 can trap the beta subunit in an inactive complex reducing the stimulation of casein phosphorylation caused by addition of beta to wild-type alpha. This competitive effect depends on the ratio of alpha/alphaA156 and on the amount of beta available. Since beta inhibits the phosphorylation of calmodulin by CK2alpha, the addition of CK2alphaA156, in this case, increases calmodulin phosphorylation by the alpha and beta combination. These results suggest that CK2alphaA156 may be a useful dominant-negative mutant that can serve to explore the multiple functions of CK2beta.

The recombinant alpha isoform of protein kinase CK1 from Xenopus laevis can phosphorylate tyrosine in synthetic substrates.

The cDNA coding for protein kinase CK1 alpha has been cloned from a Xenopus laevis cDNA library. The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38874 Da. The sequence is identical to that of the human CK1 alpha and to the bovine CK1 alpha, except that it is 12 amino acids longer than the latter protein. Southern blotting with a 264-bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DNA with EcoR1 and Hind3, suggesting that X. laevis possesses a family of related CK1 genes. CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of its characteristics were determined. The recombinant GT-CK1 alpha fusion protein was found to have apparent Km values for ATP (12 microM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 microM) which are similar to those of the rat liver CK1 enzyme. The recombinant CK1 alpha activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80:Tyr20). This inhibition is competitive and shows an approximate K1 of 5 microM. CK1 alpha can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE. This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine.

Cloning, expression and properties of the alpha' subunit of casein kinase 2 from zebrafish (Danio rerio).

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (alpha and/or alpha') and regulatory subunits (beta 2). The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter. There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha', and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263-273). In the present work, the isolation and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis. The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1). The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity. The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) and alpha (X. laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.

The casein kinase 1 alpha gene of Drosophila melanogaster is developmentally regulated and the kinase activity of the protein induced by DNA damage.

We report the molecular cloning and characterisation of the first CK1(casein kinase) gene of Drosophila melanogaster (dmCK1). The protein sequence (DMCK1) shares significant homology with other mammalian CK1 protein kinases of the alpha sub-class. The dmCK1 gene is expressed only in adult females and during early embryonic development as a single transcript. Western blot analysis of total protein extracts of different stages of development show that the gene product is likewise present during early embryogenesis and in adult females. Kinase activity studies show that DMCK1 is active when in vitro translated but inactive when immunoprecipitated from total early embryo extracts. However, after dephosphorylation treatment the immunoprecipitates show high kinase activity. More significantly, DMCK1 kinase activity present in the immunoprecipitates can be specifically activated by gamma-irradiation of early embryos. Also, when DMCK1 is immunoprecipitated after irradiation it appears to undergo phosphorylation. Immunolocalization of DMCK1 in early embryos shows that the protein is predominantly cytoplasmic but after irradiation there is a significant relocalization to the interphase nucleus. The results suggest a possible requirement of the Drosophila CK1 alpha for mechanisms associated with DNA repair during early embryogenesis.