Effectiveness of rosuvastatin plus colchicine, emtricitabine/tenofovir and combinations thereof in hospitalized patients with COVID-19: a pragmatic, open-label randomized trial.

BACKGROUND: The use of rosuvastatin plus colchicine and emtricitabine/tenofovir in hospitalized patients with SARS-CoV-2 disease (COVID-19) has not been assessed. The objective of this study was to assess the effectiveness and safety of rosuvastatin plus colchicine, emtricitabine/tenofovir, and their combined use in these patients. METHODS: This was a randomized, controlled, open-label, multicentre, parallel, pragmatic study conducted in six referral hospitals in Bogotá, Colombia. The study enrolled hospitalized patients over 18 years of age with a confirmed diagnosis of COVID-19 complicated with pneumonia, not on chronic treatment with the study medications, and with no contraindications for their use. Patients were assigned 1:1:1:1. 1) emtricitabine with tenofovir disoproxil fumarate (FTC/TDF, 200/300 mg given orally for 10 days); 2) colchicine plus rosuvastatin (COLCH+ROSU, 0.5 mg and 40 mg given orally for 14 days); 3) emtricitabine with tenofovir disoproxil plus colchicine and rosuvastatin at the same doses and for the same period of time (FTC/TDF+COLCH+ROSU); or 4) the Colombian consensus standard of care, including a corticosteroid (SOC). The primary endpoint was 28-day all-cause mortality. A modified intention-to-treat analysis was used together with a usefulness analysis to determine which could be the best treatment. The trial was registered at ClinicalTrials.gov: NCT04359095. FINDINGS: Out of 994 candidates considered between August 2020 and March 2021, 649 (65.3%) patients agreed to participate and were enrolled in this study; among them, 633 (97.5%) were included in the analysis. The mean age was 55.4 years (SD ± 12.8 years), and 428 (68%) were men; 28-day mortality was significantly lower in the FTC/TDF+COLCH+ROSUV group than in the SOC group, 10.7% (17/159) vs. 17.4% (28/161) (hazard ratio [HR] 0.53; 95% CI 0.29 to 0.96). Mortality in the FTC/TDF group was 13.8% (22/160, HR 0.68, 95% CI 0.39 to 1.20) and 14.4% in the COLCH+ROSU group (22/153) (HR 0.78, 95% CI 0.44 to 1.36). A lower need for invasive mechanical ventilation was observed in the FTC/TDF+COLCH+ROSUV group than in the SOC group (risk difference [RD] - 0.08, 95% CI 0.11 to 0.04). Three patients presented severe adverse events, one severe diarrhoea in the COLCH+ROSU and one in the FTC/TDF+COLCH+ROSU group and one general exanthema in the FTC/TDF group. INTERPRETATION: The combined use of FTC/TDF+COLCH+ROSU reduces the risk of 28-day mortality and the need for invasive mechanical ventilation in hospitalized patients with pulmonary compromise from COVID-19. More randomized controlled trials are needed to compare the effectiveness and cost of treatment with this combination versus other drugs that have been shown to reduce mortality from SARS-CoV-2 infection and its usefulness in patients with chronic statin use.

Experiencia preliminar en el tratamiento combinado de metástasis vertebrales mediante radiofrecuencia y cifoplastia en sesión única / No disponible

Objetivos: Describir nuestra experiencia preliminar en el tratamiento de metástasis vertebrales mediante radiofrecuencia y cifoplastia combinadas en sesión única. Material y métodos: Se trataron cuatro pacientes con metástasis vertebral única confirmada histológicamente (mama, próstata, pulmón y mieloma en D12, L1, L5 y D12, respectivamente). La indicación en todos los casos fue el dolor con una mala respuesta al tratamiento médico habitual. Todos los pacientes presentaban dolor en el rango 6-7 de la escala visual analógica (EVA). En dos casos existía lesión lítica del muro posterior. Tras la obtención del consentimiento informado se realizó el procedimiento bajo sedación e infiltración anestésica local. Se efectuó abordaje transpedicular bilateral con sistemas de punción ósea 11G. Se insertaron de forma coaxial dos agujas de radiofrecuencia para efectuar un ciclo de ablación por cada pedículo. Durante el ciclo de ablación la punta del dispositivo correspondiente se situó en la unión del tercio medio con el tercio anterior del cuerpo vertebral, empleando la segunda aguja como sensor térmico, con su extremo a la altura del muro posterior. La duración de cada ciclo de ablación fue de 8 minutos, alcanzando temperaturas intratumorales de 70-80 ºC. A continuación se realizó cifoplastia transpedicular. Resultados: No se registraron complicaciones intra-periprocedimiento, con alta domiciliaria en las 24 horas siguientes. En todos los pacientes hubo una mejoría inmediata del dolor tras el procedimiento (con dolor de intensidad 1-2 de la EVA). En tres pacientes se retiró progresivamente la medicación analgésica, sin evidencia en ninguno de ellos de progresión local de la enfermedad ni recurrencia-aumento del dolor en el seguimiento (dolor de intensidad 1 de la EVA en un seguimiento en el rango de 8-14 meses). En un paciente no se pudo efectuar seguimiento clínico-radiológico posterior al alta. Conclusión: El empleo de radiofrecuencia asociada a cifoplastia en la enfermedad metastásica vertebral puede contribuir al manejo del dolor refractario al tratamiento médico y al control local de la enfermedad (AU)

Objectives: Describe our preliminary experience in the treatment of vertebral metastases by radiofrequency and Kyphoplasty combined in one single session. Material and methods: Four patients with histologically confirmed single spinal metastasis (breast, prostate, lung and myeloma in L1, L5, D12, D12, respectively) were treated. The indication in all cases was pain with a poor response to medical treatment. All patients had pain in the range 6-7 visual analogue scale (VAS). In two cases there was a lytic lesion of the spinal posterior wall. After obtaining informed consent, and under sedation and local anesthetic the procedure took place. The transpedicular approach took place with a 11 G bone puncture system. Two radiofrequency needles were coaxially inserted to carry out an ablation cycle through each pedicle. During the ablation cycle the tip of the ablation neddle stood between the anterior and middle third of the vertebral body, while the second needle was used as thermal sensor with its end to the height of the vertebral posterior wall. The duration of each cycle of ablation was 8 minutes reaching intratumoral temperatures of 70-80 °C. Transpedicular Kyphoplasty was performed subsequently. Results: No complications were reported during or after the procedure and patients were discharged in the first 24 hours. There was an immediate improvement in pain after the procedure (with a VAS 1-2 intensity pain) in all patients. During follow up, analgesic medication was withdrawn in three patients, and there was no evidence of disease progression or recurrence of pain (pain intensity 1 (VAS) in a follow-up in the range of 8-14 months). Clinical and radiological follow-up after discharge could not be performed on a patient. Conclusion: The use of radio-frequency associated with Kyphoplasty in vertebral metastatic disease can contribute to the management of refractory pain to medical treatment (AU)

Cytotoxic mechanism of the ribotoxin alpha-sarcin. Induction of cell death via apoptosis.

Alpha-sarcin is a ribosome-inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of alpha-sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for alpha-sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. alpha-Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with alpha-sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase-3-like activity and cleavage of poly(ADP-ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of alpha-sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive alpha-sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z-VAD-fmk does not suppress protein synthesis inhibition by alpha-sarcin. Together, these data suggest that alpha-sarcin-induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.

African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in mammalian cells.

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

Ceramide-induced cell death is independent of the Fas/Fas ligand pathway and is prevented by Nur77 overexpression in A20 B cells.

The role of ceramide in triggering apoptosis is still a matter of debate. While in some experimental systems, ceramide was shown to mediate Fas-induced cell death, in other instances it was claimed to induce the expression of Fas ligand (FasL), killing cells in a caspase-dependent fashion. We found that, in mature A20 B cells, ceramide-induced apoptosis is independent of the caspase pathway, since we observed no ICE-like, CPP32-like and Mch2 activities and no PARP proteolysis. Moreover, we were unable to protect these cells from ceramide-induced apoptosis using caspase inhibitors, while they blocked Fas-induced apoptosis and no FasL induction could be detected following ceramide treatment. These results suggest that ceramide does not induce apoptosis through the Fas/FasL pathway. We also found that overexpression of Nur77, a zinc-finger transcription factor described to upregulate FasL, antagonizes ceramide-induced apoptosis, but not Fas-induced apoptosis. This further supports the hypothesis that Fas and ceramide death pathways are independent in A20 cells. Ceramide-induced cell death was associated with increased c-myc, p53, Bax and p27kip1 levels; in contrast, cells transfected with Nur77 (A20Nur77), resistant to ceramide-induced apoptosis, showed a marked downregulation of p53 after ceramide treatment, with neither Bax nor p27kip1 induction. In conclusion, our results suggest that, in the A20 B cell line, Fas and ceramide trigger two distinct pathways and that Nur77 overexpression confers protection against ceramide-mediated apoptosis which correlates with inhibition of p53, Bax and p27kip1 induction.

Adenovirus lacking the 19-kDa and 55-kDa E1B genes exerts a marked cytotoxic effect in human malignant cells.

UNLABELLED: The adenovirus (Ad) E1A gene exerts an antitumor effect and can induce sensitivity to treatment with DNA-damaging agents. In contrast, the Ad 19-kDa E1B protein inhibits E1A-mediated apoptosis and the 55-kDa E1B inactivates the p53 protein. In this paper, we study the in vitro and in vivo effects of a 19-kDa and 55-kDa E1B-defective Ad in several malignant human tumor cell lines. MATERIALS AND METHODS: Nontumorigenic human fibroblasts (CCD-45SK and Hs67), peripheral blood lymphocytes, and several human tumor cell lines derived from cervix, colon, and breast carcinomas, epidermoid carcinoma, and osteosarcoma (HeLa, HT29, MCF7, Saos-2, and A431 cell lines) were studied. Wild-type (wt) Ad type 5 and H5 dL118 Ad, a mutant with the deleted E1B region, were employed. The cells were infected at 20 plaque-forming units, and cell viability was evaluated by the crystal violet method. In the in vivo experiments, 2 x 10(6) cells from the carcinoma cell lines HeLa, A431 and HT29 were injected into nude mice. The tumorigenicity of previously infected cells and after an intratumoral injection of Ad was analyzed. The mice received whole-body gamma-irradiation. RESULTS: The H5 dL118 mutant produced a marked cytopathic effect in all of the malignant cells, surpassing that of the wt Ad; viability at 72 hours ranged from 11% to 20% for H5 dL118 Ad and from 70% to 93% for the wt Ad with respect to uninfected controls. In the in vivo experiments, a total inhibition of tumorigenicity was detected when cells were infected prior to injection and a partial and transitory decrease in tumorigenicity was detected when the mutant H5 dL118 was injected intratumorally. gamma-irradiation enhanced the in vivo antitumor effects. CONCLUSIONS: These results indicate that infection with completely E1B-deficient Ads induced a marked cytopathic effect on malignant cells that was higher than that seen for wt Ads; in addition, infection with such Ads exerts a tumor suppressor effect in vivo.

Implication of calpain in caspase activation during B cell clonal deletion.

In the absence of costimulating signals, B cell receptor (BCR) crosslinking on immature B cells triggers the apoptotic cell death program. In the WEHI-231 B cell lymphoma model, anti-IgM crosslinking triggers activation of caspase-7 independently of caspase-8, followed by apoptosis. Two main mechanisms for caspase-7 activation have been proposed: (i) caspase-8 recruitment to death receptors (Fas or tumour necrosis factor); and (ii) changes in mitochondrial membrane permeability and cytochrome c release, which activate caspase-9. Here we report that caspase-7 activation induced by BCR crosslinking is independent of caspase-8 and cytochrome c translocation from mitochondria to the cytosol, as well as of mitochondrial depolarization. In addition, in a cell-free system, the S-100 fraction of anti-IgM-treated WEHI-231 cells induces a caspase activation pattern different from that activated by cytochrome c and dATP. We demonstrate that calpain specifically triggers activation and processing of caspase-7 both in vitro and in vivo, and that both processes are inhibited by calpain inhibitors. Furthermore, calpain activation is associated with decreased expression levels of calpastatin, which is upregulated by CD40 ligation. These data confirm a role for calpain during BCR crosslinking, which may be critical for cell deletion by apoptosis during B cell development and activation.

Caspase activation by BCR cross-linking in immature B cells: differential effects on growth arrest and apoptosis.

The B cell lymphoma WEHI-231 has been used as a model to study immature B cell tolerance, based on its capacity to undergo growth arrest and programmed cell death on B cell receptor (BCR) cross-linking. Using this model to identify the molecular mechanisms underlying these processes, we found that BCR cross-linking results in the selective activation of caspase 7/Mch3, but not of the other two members of the CPP32 family, caspase 2/Nedd2 and caspase 3/CPP32. This was evidenced by the induction of proteolytic activity against the substrate for the CPP32 subfamily of caspases (z-DVED-AMC) in vitro, as well as PARP proteolysis in vivo and by the processing of the 35 kDa Mch3 into a 32 kDa species, which was later further proteolyzed. The general caspase inhibitor z-VAD-fmk, but not the CPP32 family inhibitor Ac-DEVD-CHO, blocked anti- micro-induced apoptosis, indicating that a caspase not belonging to the CPP32-like family is also implicated in anti- micro-triggered apoptosis. In contrast, z-VAD-fmk was not able to counteract growth arrest induced by anti- micro treatment, suggesting that caspase activation is not necessary for induction of growth arrest. Neither of the inhibitors prevented Mch3 processing; however, z-VAD-fmk prevented proteolysis of the p32 subunit, suggesting that further processing of this subunit is associated with apoptosis. Bcl-2 overexpression prevented anti- micro induction of CPP32-like activity and apoptosis, and blocked further processing of the Mch3 p32 subunit. In contrast, CD40 stimulation completely blocked the appearance of the p32 subunit in addition to blocking CPP32-like activity and apoptosis induced by BCR cross-linking. Moreover, only CD40 stimulation was able to prevent anti- micro-induced growth arrest, which was correlated with inhibition of retinoblastoma and of cyclin A down-regulation. In splenic B cells, Mch3 is also specifically proteolyzed ex vivo after induction of apoptosis by BCR cross-linking, demonstrating the specific involvement of caspase-7/Mch3 in apoptosis induced in B cell tolerance.

Blocked negative selection of developing T cells in mice expressing the baculovirus p35 caspase inhibitor.

Clonal deletion in the thymus by apoptosis is involved in purging the immune system of self-reactive T lymphocytes (negative selection). Cysteine proteases (caspases) belonging to the CPP32 family are activated during this process. We have produced transgenic mice expressing baculovirus p35, a broad-range caspase inhibitor. Thymocytes from p35 transgenic mice were resistant in vitro to several apoptosis-inducing agents; this resistance correlated with the inhibition of CPP32-like activity. Negative selection in vivo of thymocytes triggered by two exogenous antigens, staphylococcal enterotoxin B superantigen and an antigenic peptide in the F5 T-cell receptor transgenic model, was specifically inhibited in p35 transgenic mice. Our results provide direct evidence for caspase involvement in negative selection during thymocyte development.

SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.

We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.

Prolonged half-life in the circulation of a chemical conjugate between a pro-urokinase derivative and human serum albumin.

Pro-urokinase is a natural plasminogen activator that displays a clot-lysis activity through a fibrin-dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue-type plasminogen activator and two-chain urokinase-type plasminogen activator, pro-urokinase has a very short half-life in circulation. It has been described that conjugation of serum albumin with pro-urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N-terminal end of des-(C11-K135)-pro-urokinase (delta 125-proUK), a pro-urokinase deletion mutant lacking amino acids 11-135. We have expressed and purified the new mutein [H5K, S9C, N10T] des-(C11-K135)-pro-urokinase (Cys-delta 125-pro-urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys-delta-pro-urokinase. The purified conjugate obtained has a lower specific amidolytic activity (72,000 U/mg) than unconjugated Cys-delta 125-pro-urikinase (240,000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of delta 125-pro-urokinase. We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half-life in the circulation, with respect to pro-urokinase and delta 125-pro-urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro-urokinase.

Dermatomicosis en población de Manizales / Dermatomycosis in population of Manizales

Se informa sobre el estudio micológico realizado en una población de 1.175 pacientes adultos remitidos por el servicio médico de la ciudad de Manizales. Fue posible establecer diagnóstico positivo en 779 pacientes (66,3 por ciento), con 72 (6,1 por ciento) casos de pitiriasis versicolor, 1(0,1 por ciento) por Tinea nigra palmaris, 291 (24,8 por ciento) micosis cutáneas por dermatofitos y 184 (15,7 por ciento) casos de candidiasis. La distribución de frecuencia agrupó a 15 Microsporum, 101 Epidermophyton y 257 Trichophyton. Fueron 357 (95,7 por ciento) antropofílicos, 10 (2,7 por ciento) geofílicos y 6 (1,6 por ciento) zoofílicos. Las especies aisladas fueron: M canis, M. gypseum, E. floccosum, T. rubrum, T. mentagrophytes, T. tonsurans, T. verrucosum, Cladosporium werneckii, Malassezia furtur y otros. En el estudio de las onicopatías se encontraron: 82 (7,0 por ciento) casos por dermatofitos 103 (8,8 por ciento) casos por Candida albicans; en 46 (3,9 por ciento) casos se aislaron hongos no dermatófitos incriminados como agentes patógenos oportunistas, por algunos autores. No hubo diagnóstico micológico en 10 (0.8 por ciento) casos por obtenerse en los cultivos Mycelia sterilla, y fueron negativos 386 (32,8 por ciento). Se indican los procedimientos para análisis micológico y se mencionan consideraciones generales sobre la patología de la dermatofitos

Production and secretion of human interleukin 6 into the periplasm of Escherichia coli: efficient processing of N-terminal variants of hIL6 by the E. coli signal peptidase.

We have developed a system for expressing human interleukin 6 into the periplasmic space of Escherichia coli. The method is based on the expression of the hIL6 gene under the control of the regulatory signals of plasmid pINIII-OMPA3, i.e., lpp-lac promoter and the E. coli OMPA ribosome binding site and leader sequence. Since microheterogeneity is known to occur in the amino end of the cytokine, we tested different 'natural' versions of the protein, and we found that the secretion process was only efficient when the N-terminal amino acid was not proline. In flask experiments this procedure yields about 8-10 mg of biologically active hIL6 per liter.

Enzymatic synthesis of penicillins.

Different penicillins (phenylacetyl, 2-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, phenoxyacetyl and 2-thiopheneacetylpenicillin) have been synthesized "in vitro" by direct N-acylation of 6-aminopenicillanic acid (6-APA) with the acyl group of several acyl-CoA derivatives. The enzyme that catalyzes these reactions, acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum, was purified to homogeneity (374-fold) and its amino acid composition is given. This protein accepts as substrates several aliphatic acids and different aromatic acids with the only requirement that an acetyl-CoA moiety must be present in the substrate molecule. Shortening or lengthening of the acyl moiety prevents the 6-APA-N-acylation reaction. The presence of an amino group in the alpha-position of the acetyl group does not allow this molecule to be used as substrate. However, different substitutions in the phenyl group (hydroxylation of the carbons 2 and 4) or its replacement by another aromatic ring (thiophene) were accepted with varying reactions rates in the acylation reaction when a 176-fold purified acyltransferase was employed. The homogeneity pure enzyme accepts as substrate thiophene acetyl-CoA but it did not 2-hydroxyphenyl and 4-hydroxyphenylacetyl-CoA. The presence of an oxygen atom between the aromatic and the acetyl moieties did not affect the catalysis.

Structure of the pigeon lysozyme and its relationship with other type c lysozymes.

1. The secondary structure of the pigeon egg-white lysozyme shows important differences when compared to other type c lysozymes. These differences are mainly located at the region comprising residues 77-84. This segment contains one alpha-helix in the lysozymes c studied by means of an X-ray analysis, while the residues at such positions in pigeon lysozyme would form two beta-bends. 2. Analysis of the tertiary structure of the pigeon lysozyme by means of hydropathy profiles reveals that the above segment seems to be more hydrophilic in the pigeon enzyme than in other type c lysozymes. 3. Though a certain similarity to the calcium-binding loop of alpha-lactalbumins is detected in pigeon lysozyme, the circular dichroism spectra of the protein at neutral pH do not change in the presence of Ca2+ ions. 4. The presented structural analysis is discussed in terms of function-structure and antigenicity relationships between the type c lysozymes.

Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila.

The penicillin acylase (PAC) from Kluyvera citrophila ATCC21285 has been purified to homogeneity and found to be composed of two non-identical subunits of 23 and 62 kDa, in contrast with the previous findings [Shimizu et al., Agr. Biol. Chem. 39 (1975) 1655-1661]. The nucleotide (nt) sequence of the K. citrophila pac gene contained in the 3-kb PvuI-HindIII fragment of pKAP1 [García and Buesa, J. Biotechnol. 3 (1986) 187-195] has been determined, showing that it encodes a protein of 844 amino acid (aa) residues. The aa analysis of the N-terminal and C-terminal sequences of the purified subunits showed that they were derived from a common precursor protein of 93.5 kDa, from which a signal peptide of 26 aa, responsible for the periplasmic translocation of the protein, and an internal connecting polypeptide of 54 aa, have been removed in the maturation of the PAC. The comparison of the nt sequences of the pac genes from K. citrophila and Escherichia coli ATCC11105 [Schumacher et al., Nucl. Acids Res. 14 (1986) 5713-5727] revealed 80% homology, suggesting a common ancestral pac gene origin. The results reported here should allow investigation of the unusual mechanism of maturation of this prokaryotic protein, as well as manipulation, using DNA recombinant techniques, of the catalytic properties of this industrially important enzyme.

Amino acid sequence of pigeon egg-white lysozyme.

The amino acid sequence of pigeon egg-white lysozyme has been determined. The protein molecule contains a single polypeptide chain of 127 amino acid residues and exhibits only about 60% homology when compared to hen egg-white lysozyme.

[Hyperproduction of L-proline in Escherichia coli]. / Hiperproducción de L-prolina en Escherichia coli.

The proA and proB genes from Escherichia coli have been cloned using the plasmids pBR322 and pBR325 as vectors. The episome F128 (proAB, lac) was used as cloning DNA source. Both genes were firstly located within a 10 kilobases EcoRI DNA fragment of the episome. Subcloning experiments showed that both proteins were coded by a 3 kilobases PstI DNA fragment. Although, th recombinant plasmids containing the proA and proB genes were able to complement the Pro- phenotype of different E. coli strains, bacteria harboring these plasmids did not excrete L-proline to the culture medium. Nevertheless, an operon, proAB, able to confer to E. coli cells the property of excreting L-proline, was isolated from an UV-mutant of E. coli E5014 [F' (proAB, lac)] resistant to the L-proline analogue, thioproline. E. coli HB101 cells transformed with the plasmid pJABP (UV) carrying the mutated proAB operon excreted up to 5 g/l of L-proline, after 40 hours of fermentation at 37 degrees C in a modified M63 minimal medium. The production of L-proline was not increased when the proC gene was inserted in the plasmid pJABP (UV).