Base excision repair proteins couple activation-induced cytidine deaminase and endonuclease G during replication stress-induced MLL destabilization.

The breakpoint cluster region of the MLL gene (MLLbcr) is frequently rearranged in therapy-related and infant acute leukaemia, but the destabilizing mechanism is poorly understood. We recently proposed that DNA replication stress results in MLLbcr cleavage via endonuclease G (EndoG) and represents the common denominator of genotoxic therapy-induced MLL destabilization. Here we performed a siRNA screen for new factors involved in replication stress-induced MLL rearrangements employing an enhanced green fluorescent protein-based reporter system. We identified 10 factors acting in line with EndoG in MLLbcr breakage or further downstream in the repair of the MLLbcr breaks, including activation-induced cytidine deaminase (AID), previously proposed to initiate MLLbcr rearrangements in an RNA transcription-dependent mechanism. Further analysis connected AID and EndoG in MLLbcr destabilization via base excision repair (BER) components. We show that replication stress-induced recruitment of EndoG to the MLLbcr and cleavage are AID/BER dependent. Notably, inhibition of the core BER factor Apurinic-apyrimidinic endonuclease 1 protects against MLLbcr cleavage in tumour and human cord blood-derived haematopoietic stem/progenitor cells, harbouring the cells of origin of leukaemia. We propose that off-target binding of AID to the MLLbcr initiates BER-mediated single-stranded DNA cleavage, which causes derailed EndoG activity ultimately resulting in leukaemogenic MLLbcr rearrangements.

Endonuclease G initiates DNA rearrangements at the MLL breakpoint cluster upon replication stress.

MLL (myeloid/lymphoid or mixed-lineage leukemia) rearrangements are frequent in therapy-related and childhood acute leukemia, and are associated with poor prognosis. The majority of the rearrangements fall within a 7.3-kb MLL breakpoint cluster region (MLLbcr), particularly in a 0.4-kb hotspot at the intron11-exon12 boundary. The underlying mechanisms are poorly understood, though multiple pathways including early apoptotic signaling, accompanied by high-order DNA fragmentation, have been implicated. We introduced the MLLbcr hotspot in an EGFP-based recombination reporter system and demonstrated enhancement of both spontaneous and genotoxic treatment-induced DNA recombination by the MLLbcr in various human cell types. We identified Endonuclease G (EndoG), an apoptotic nuclease, as an essential factor for MLLbcr-specific DNA recombination after induction of replication stress. We provide evidence for replication stress-induced nuclear accumulation of EndoG, DNA binding by EndoG as well as cleavage of the chromosomal MLLbcr locus in a manner requiring EndoG. We demonstrate additional dependency of MLLbcr breakage on ATM signaling to histone H2B monoubiquitinase RNF20, involved in chromatin relaxation. Altogether our findings provide a novel mechanism underlying MLLbcr destabilization in the cells of origin of leukemogenesis, with replication stress-activated, EndoG-mediated cleavage at the MLLbcr, which may serve resolution of the stalled forks via recombination repair, however, also permits MLL rearrangements.

High testosterone levels of ovarian origin affect adrenal steroidogenesis?

Androgens of ovarian origin have been suggested to affect adrenal enzymatic activity. To investigate this possibility, the 17-hydroxyprogesterone (17-OH P) and cortisol (F) responses to an ACTH stimulation test (0.25 mg iv, bolus) were evaluated in 10 normal women and in 39 hyperandrogenic women with normal (14 subjects) or high (25 subjects) testosterone (T) levels. The 17-OH P release and the ratio between 17-OH P and F release in response to the ACTH stimulation test were significantly higher (P less than 0.05) in hyperandrogenic women with high T levels than in normal subjects. Eight hyperandrogenic women with high T received intranasal GnRH agonist (Buserelin, 1200 micrograms/day) for 4 weeks, and the 17-OH P and F release in response to the ACTH stimulation was reassessed after agonist treatment. At the end of GnRH agonist administration the mean circulating levels of T were significantly reduced (P less than 0.05). The F response to the ACTH test was not modified by pretreatment with the GnRH agonist. The 17-OH P response to the ACTH stimulation test after the GnRH agonist was unchanged in comparison with control tests, as well as the ratio between 17-OH P and F responses to the ACTH test. These data do not seem to confirm, as previously suggested, that high T levels of ovarian origin affect adrenal steroidogenesis.

Topical treatment of burn wounds with chloroxidating solution and silver sulfadiazine: a comparative study.

The present paper reports the results of clinical and laboratory tests carried out on two homogeneous groups of ten burn patients subjected to local therapy, either with isotonic chloroxidating solution Amuchina* or with 1% silver sulfadiazine cream at the Burns Centre of the Pisa University Dermatological Clinic. The local systemic behaviour of the patients examined was evaluated for the containment of septic complications at the burn site. In the group subjected to treatment with chloroxidating solution, sepsis appeared to have a lower incidence in the evolution of dermatitis in the phase of escharolysis, in the formation of granulation tissue, and in the attachment of cutaneous grafts. The systemic involvement (temperature curve, etc.) appeared to be more marked for some patients treated with silver sulfadiazine in response to septic aggression of the burn wounds. On the basis of data referring to the development of the wound granulation and the temperature curve, as well as the microbial presence and the subjective tolerance of the medication, the comparison was favourable, making all necessary allowances, to topical treatment with electrolytic chloroxidating solution; other comparative data were at the limit of significance.

[Anthocyanosides and the walls of the microvessels: further aspects of the mechanism of action of their protective effect in syndromes due to abnormal capillary fragility]. / Antocianosidi e parete dei microvasi nuovi aspetti sul modo d'azione dell'effetto protettivo nelle sindromi da abnorme fragilità capillare.

On the basis of previous biochemical observations, which have demonstrated the formation of complexes between anthocyanosides and some phospholipids, the AA. investigate the modifications induced by local and general administration of anthocyanosides 1) on the foreign body granuloma and 2) on the composition of the protein fractions in the exudate from the capillaries of the granulation tissue, growing on post-thromboflebitic or varicose leg ulcerations. The biochemical and histochemical data may show that the anthocyanosides protect the altered capillary walls with a double mechanism: a) increasing the endothelium barrier-effect through a stabilisation of the membrane phospholipids and b) increasing the biosynthetic processes of the acid mucopolysaccharides of the connective ground substance, by restoring the altered mucopolysaccharidic pericapillary sheat. This last effect may explain the marked increase of new-formed capillaries and collagen fibrils induced by the anthocyanosides.

Collagen as a pharmacological approach in wound healing.

The authors have reviewed the most important biological mechanisms involved in wound healing, the main agents that modify the healing process and the physiological and pharmacological role of collagen. Putative mechanisms of collagen in wound repair are described with particular emphasis on haemostatic effect, interaction with platelets and fibronectin, properties of of increasing fluid exudate and its cellular component (macrophages) and the "scaffold" role for fibroblastic proliferation. Experimental and clinical data clearly suggest that the potential use of collagen in wound repair and its main therapeutical applications: treatment chronic leg ulcers and pressure sores, burns, urological surgery, gynaecological surgery, dentistry and oral surgery, reconstructive surgery, abdominal and vascular surgery, orthopaedy.

Lyophilized type-I collagen and chronic leg ulcers.

Lyophilized type I collagen (L.C.) can stimulate wound healing by recruiting a number of different cell types (i.e. platelets and macrophages) and proteins (i.e. fibronectin). Platelets and macrophages produce locally-acting growth factors that in turn induce fibroblast and epidermal migration, angiogenesis and increase matrix synthesis. Chronic leg ulcers (C.L.U.) are the end result of microvascular failure owing to ischemia and stasis. When L.C. has been used in the treatment of C.L.U. we have observed that: a) it is significantly more effective in stimulating the healing of chronic venous ulcers when compared to hydrocolloids (p less than .05), the two products being applied upon half of the same ulcer; b) in the treatment of C.L.U. due to arterial obstruction L.C. is more effective than hydrocolloids without achieving statistical significance; c) it is very effective in the treatment of C.L.U. in thalassaemic patients; d) telethermographic studies have demonstrated an increase of blood perfusion and histological studies have shown the stimulation of angiogenesis, fibropoiesis and epidermal growth; e) the application of L.C. determines the maximum obtainable increase also under conditions of proven cicatrization difficulty; and f) enzymatic degradation of L.C. has not promoted any bacterial infection and no local or generalized sensibilization phenomena have been observed. We can conclude that L.C. is a pharmacological approach to wound healing, directly interfering with cellular and non-cellular components, and significantly improves the reparative process when delayed.

Healing of open skin surfaces with collagen foils.

Applications of Condress (patented sheets of pure bovine collagen) on open skin surfaces (30 cases of "ulcus cruris", 5 cases of decubitus, and malum perforans; 10 cases of full-thickness burns) were examined in a controlled trial. Quantification of regeneration speed, macrophotographic survey of granulation tissue and epithelial border, thermographic and chromometric evaluation of the skin microcirculation, and histological observation of regenerating tissues, were the parameters used. The following results were obtained: marked reduction of healing time, different aspects of the granulation-tissue responses, different times of topical collagenolysis, increased vascular perfusion, histological activation of angiogenesis, fibrogenesis, histiomacrophage function and superficial absorption. The employment of Condress in burn areas seems to be highly promising.

[The use of intravenous immunoglobulins in patients with severe burns]. / Uso endovenoso delle immunoglobuline nell'ustionato grave.

The parenteral use of whole molecule immunoglobulins is an important instrument for immunotherapy which can be employed in a variety of morbid conditions characterized by immunodeficiency, and therefore also in the management of severe burns. In order to confirm this assumption, the effect of i.v. administration of immunoglobulins has been studied in a group of burn patients and has been compared with the results obtained in the same number of clinically homologous patients not submitted to this treatment. In view of the results obtained, i.v. immunoglobulins can be usefully employed for the control of inflammatory and septic phenomena with reduction of abnormal parameters and a positive influence on the recovery curve.

[The relationship between achievement of a simple movement and changes in the individual components of an evoked potential]. / Izmeneniia otdel'nykh komponentov vyzvannogo potentsiala v sviazi s vypolnemiem prostogo dvizheniia

Various moments of preparation and performance of a simple voluntary movement were tested in man with the aid of somatosensory, auditory, and visual evoked responses. An obvious attenuation of somatosensory responses closely related to the movement but independent of the spontaneous EEG changes and unaffected by the ischaemic deafferentation of the active limb, was observed. The time course of the amplitude changes was different for separate components of the same evoked response. The evoked response to stimulation of the inactive limb and the auditory evoked response changed also, while the visual evoked response remained unchanged. The described changes cannot be explained by changes in the transmission via the specific pathways. The changes in the response early components are supposed to manifest facilitation or activation of the sensory-motor cortical neurons, and those of the late, generalized complex--a decrease in the ascending message from unspecific structures.

The suppressive activity of T-lymphocytes and serum factors in burned patients.

This study was performed to investigate the cell-mediated immune response in burned patients with no septic episodes. The results show that burned patients with percentage body burn higher than 20 had an impaired lymphocyte reactivity to phytohaemagglutinin and conconavalin A. This hyporesponsiveness appeared on day 3-4 and in all cases reached its maximum on day 7-8 post burn, while recovery occurred between day 11 and 29 depending on the severity of the injury. The serum from immunodepressed patients was able to inhibit the response to phytohaemagglutinin and conconavalin A of normal lymphocytes. This immunosuppressive activity was present very early after injury (on day 1-2) and before the onset of lymphocyte hyporesponsiveness to mitogens and was no longer detectable on day 7-8 post burn, when patient lymphocytes showed the greatest hyporesponsiveness to mitogens. This late depression was due to T suppressor cells.

[The relationship between strength and the amplitude of evoked response components in the human somatosensory cortex]. / Zavisimost' amplitudy komponentov vyzvannogo otveta somato-sensornoi zony kory cheloveka ot sily razdrazheniia

The intensity curves were determined for the individual components of the human somatosensory evoked response, led off from the contra and ipsilateral motor and contralateral visual areas. The amplitude of the early components steeply increased, reached "saturation" and decreased with more intensive stimuli, their peak latencies being constant. The late components were characterised by continuous increase of amplitude, and the decrease of peak latencies for the lowest part of the curve. The responses in both frontal regions were identical, while those from the frontal and posterior leads were significantly different. The obtained data indicate a marked difference between mechanisms producing individual components of the evoked response.