1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10.

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.

Immune escape of melanoma: first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway.

Sequence analyses of the transporter associated with antigen processing (TAP) in tumor cell lines with deficient MHC class I surface expression identified a bp deletion at position 1489 near the ATP-binding domain of Tap1, causing a frameshift in one melanoma cell line. The impaired TAP1 protein expression was associated with deficient TAP2 protein expression, peptide binding, translocation, and MHC class I surface expression. Stable TAP1 gene transfer reconstitutes the described defects, whereas lysis by HLA-A2-restricted CTLs was still abrogated. This was attributable to a 2-bp insertion at position 890 in the HLA-A2 gene and was corrected after HLA-A2 cotransfection. This study describes for the first time mutations in two distinct components of the MHC class I antigen processing pathway, suggesting an immune selection against CTLs recognizing both TAP-dependent and -independent T-cell epitopes.

Function of the transport complex TAP in cellular immune recognition.

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility complex (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. The MHC-encoded ABC transporter works in concert with the proteasome and MHC class I molecules for the antigen presentation on the cell surface for T cell recognition. TAP forms a heterodimer where each subunit consists of a hydrophilic nucleotide binding domain and a hydrophobic transmembrane domain. The transport mechanism is a multistep process composed of an ATP-independent peptide association step which induces a structural reorganization of the transport complex that may trigger the ATP-driven transport of the peptide into the endoplasmic reticulum lumen. By using combinatorial peptide libraries, the substrate selectivity and the recognition principle of TAP have been elucidated. TAP maximizes the degree of substrate diversity in combination with high substrate affinity. This ABC transporter is also unique as it is closely associated with chaperone-like proteins involved in bonding of the substrate onto MHC molecules. Most interestingly, virus-infected and malignant cells have developed strategies to escape immune surveillance by affecting TAP expression or function.

Phase II study of doxifluridine in advanced colorectal adenocarcinoma.

Doxifluridine, a new fluoropyrimidine derivative, was tested in a cooperative phase II trial by the Swiss Group for Clinical Cancer Research in advanced measurable colorectal cancer. The drug was given in a five consecutive day schedule by a bolus intravenous injection at a dose of 4 g/m2 per day and repeated every three to four weeks. Of 42 eligible patients, 40 had no previous chemotherapy. Response was defined in 27 patients having received two or more courses of doxifluridine. Seven responses (26%) were observed. Responses were seen only in sigmoid and rectum primary tumors. Toxicity consisted mainly of leukopenia (53% of the evaluable patients), nausea and vomiting (38%). Other toxicities such as dermatitis, myocardial injury, and hair loss were also observed. Doxifluridine has therapeutic activity, albeit limited, in advanced rectosigmoid adenocarcinoma.

Enhanced expression of human ABC-transporter tap is associated with cellular resistance to mitoxantrone.

Multidrug resistance (MDR) phenotypes have been associated with the overexpression of various members of the superfamily of ATP binding cassette (ABC) transporters. Here we demonstrate that a member of the ABC-transporter family, the heterodimer 'transporter associated with antigen processing' (TAP), physiologically involved in major histocompatibility complex class I-restricted antigen presentation, is significantly overexpressed in the human gastric carcinoma cell line EPG85-257RNOV exhibiting a mitoxantrone-resistant phenotype. This tumor cell line shows an atypical MDR phenotype in the absence of 'P-glycoprotein' or 'MDR-associated protein' overexpression but with an enforced 'breast cancer resistance protein' expression level. Transfection of both TAP subunits encoding cDNA molecules, TAP1 and TAP2, into the drug-sensitive parental gastric carcinoma cell line EPG85-257P conferred a 3.3-fold resistance to mitoxantrone but not to alternative anti-neoplastic agents. Furthermore, cell clones transfected with both, but not singularly expressed TAP1 or TAP2, reduced cellular mitoxantrone accumulation. Taken together, the data suggest that the heterodimeric TAP complex possesses characteristics of a xenobiotic transporter and that the TAP dimer contributes to the atypical MDR phenotype of human cancer cells.

Long-term results of renal transplantation in recipients with a functioning graft for 2 years.

The late results of renal transplantation are reviewed in 214 recipients with a functioning allograft for 2 years. Graft survival was better (P less than 0.001) in living related recipients (t 1/2 = 17 years) compared with cadaver graft recipients (t 1/2 = 7.7 years). Graft survival was also significantly different (P less than 0.001) in patients with a 2-year serum creatinine level of less than or equal to 2.0 (t 1/2 = 16.4 years), 2.1 to 3.0 (t 1/2 = 6.5 years), or greater than 3.0 mg/dl (t 1/2 = 2.9 years). A greater proportion of patients with a 2-year serum creatinine level of greater than 3 mg/dl had experienced greater than two rejection episodes (P less than 0.0001). Among recipients with a 2-year serum creatinine level of less than or equal to 2.0 mg/dl, living related grafts achieved better graft survival than cadaver grafts (P less than 0.05). Major complications of transplantation were more common in patients with a cadaver graft, 2-year serum creatinine level of greater than 3 mg/dl, or age greater than 45 years. One hundred and forty-two patients are currently alive, 93% of whom have achieved complete rehabilitation.

Modification of Marshall-Marchetti-Krantz operation.

In a classic article published in 1949 Marshall, Marchetti, and Krantz demonstrated that stress incontinence in women without uterine prolapse could be corrected by a simple vesicourethral suspension. Beginning in 1960 one of the authors (W.E.C.) became concerned about suturing the urethra to the periosteum of the pubis. It occurred to him that use of the upper sutures only to pull up the bladder and hold its anterior wall to the back of the rectus muscles might be just as effective, and this has proved to be so.

Phase I study of cyclohexylamine and lysine salt of mafosfamide.

Mafosfamide is a new oxazaphosphorine that breaks down spontaneously into 4-hydroxy-cyclophosphamide. A phase I trial with cyclohexylamine and lysine salts of mafosfamide was carried out in 16 patients, using weekly IV perfusion. Dose-limiting toxicities were not hematological, but consisted in the development of severe pain along the vein during administration. A particular mucosal syndrome with sneezing and conjunctivitis was seen only after administration of the lysine salt. The dose of 700 mg/m2 per week represents the maximum tolerated dose with this weekly schedule.

N-(Phosphonacetyl)-L-aspartate (PALA): current status.

N-(Phosphonoacetyl)-L-aspartate (PALA) is a synthetic antimetabolite exhibiting striking oncolytic properties against a wide variety of experimental solid tumors. The clinical dose-limiting factor is mucocutaneous toxicity which is reversible and dose-related. Delineation of the single-agent activity of PALA in human cancer must still await results of recently activated trials.

Cisplatin: impact of a new anticancer agent on current therapeutic strategies.

This brief overview illustrates the role of cisplatin in current cancer chemotherapy. This new agent is increasingly utilized in first line regimens for a variety of tumor types notably testicular, head and neck and bladder cancer. Large scale investigations are taking place to evaluate these regimens in combined modality approaches to early stages of these diseases.

Characteristic pattern of free amino acids in plasma and skeletal muscle in stable hepatic cirrhosis.

Free amino acid (AA) concentrations in plasma and quadriceps femoris muscle were determined in 19 healthy volunteers and in 16 patients with hepatic cirrhosis and portal hypertension. Nutritional state was impaired as judged by overt muscle wasting (9/16), triceps skinfold thickness less than 70% of normal in 8/14 (57%), and creatinine-height index below 70% in 5/12 (42%). In the plasma of patients the typical amino acid pattern of cirrhosis was to be observed: Elevation of tyrosine and methionine (p less than 0.01), uniform reduction of branched chain amino acids (p less than 0.001) resulting in a decreased molar ratio of BCAA/AAA from 2.85 +/- 0.05 in normal individuals to 1.35 +/- 0.12 in cirrhotics (p less than 0.001). Levels of the gluconeogenic AA glutamine, glutamate, aspartate, alanine, glycine, threonine, serine and lysine were lowered (p less than 0.05). In muscle of cirrhotics, intracellular AA concentrations exhibited a similar pattern with two major exceptions: Tyrosine and phenylalanine were augmented (p less than 0.001). Surprisingly, BCAA levels were altered heterogeneously; those of gluconeogenic BCAA decreased: Valine from 0.34 +/- 0.03 to 0.20 +/- 0.03 mmol/l (p less than 0.001), isoleucine 0.09 +/- 0.01 to 0.05 +/- 0.02 mmol/l. However, the concentration of ketogenic leucine remained unaltered in muscle. Nevertheless, the molar ratio of BCAA/AAA was considerably reduced from 3.70 +/- 0.04 to 0.81 +/- 0.08 (p less than 0.001). Most of the gluconeogenic AA exhibited reduced intramuscular concentrations, but glutamine levels were normal. The pattern of plasma and muscle free AA in hepatic cirrhosis is thus characterized by accumulation of aromatic AA and by depletion of gluconeogenic AA, especially BCAA.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple myeloma presenting with external ear canal mass.

The manifestations of multiple myeloma are protean and related to bony osteolytic lesions, and to medullar and renal insufficiency. We report a patient who presented with otalgia as the inaugural symptom of multiple myeloma. Local irradiation combined with systemic chemotherapy led to the disappearance of the temporal bone mass and the accompanying symptoms. To date, 24 months after the diagnosis, the patient is still in remission. The literature on otological involvement in multiple myeloma is reviewed. Symptoms are non-specific and include hearing loss, tinnitus, dizziness, facial paralysis, and otalgia. The diagnosis of multiple myeloma should be considered in the presence of a temporal bone mass.

[Histological study of a case of chondrocostal aspergillosis]. / Etude histologique d'un cas d'aspergillose chondrocostale.

Histological study of biopsy specimens successively taken from the 7th, 5th and 6th right chondrocostal regions in a 33 year-old man; the patient was under chemotherapy for a diffuse lymphoma and has been operated for an aspergilloma of the right lung superior lobe. Non-specific chronic osteomyelitis was observed as well as Aspergillus fumigatus perichondritis (acute and chronic) which had lead to cartilage fragmentation. Cartilage fragmentation could be attributed to an enzymatic chondrolysis induced by polymorphonuclears inflammation. Such matrix degradation seems to favour the penetration of aspergillus into the cartilage surface. This case report recalls that fungi (including Aspergillus despite it appears exceptional) must be considered as a possible cause of osteomyelitis, particularly in immunosuppressed patients.

[Chromosome 5q-- in the medullar cells of a patient with anaemia which later developed into acute non-differentiated leukaemia (author's transl)]. / Chromosome 5q-- dans les cellules médullaires d'un sujet atteint d'anemie ayant evolue en leucémie aigue indifferenciee.

The authors report the observation of a 76-year-old man who since 1974 had a persistent anaemia considered as a pre-leukaemic state. The patient was hospitalized in May 1977 with fever and severe asthenia. The laboratory results indicated a probable diagnosis of acute non-differentiated leukaemia of stem cells. In spite of treatment, the anaemia grew worse, the leukocytosis accompanied by blast cells became more pronounced, a massive thrombopenia occurred and the patient died in irreversible shock. Cytogenetic examination done on a medullar culture revealed the presence in all the cells of a chromosome No. 5 with the long arms deleted : 46,XY,5q--. This rare medullar anomaly was reported for the first time in 1974-1975 by the Louvain school (Van den Berghe, Sokal, et al.) in a group of refractory anaemias. It has also been described in association with other chromosomal aberrations, in anaemias or other hemopathies which all developed into acute myeloblastic leukaemia. The clinical evolution and the cytogenetic data of the patient presented here are compared with those of other cases of 5q-- published in the literature, and the significance of this 5q-- chromosome aberration in hemopathies is discussed.

[Chemotherapy of non-seminoma tumours of the testis (author's transl)]. / Chimiothérapie des tumeurs non séminomateuses du testicule.

During the past few years considerable progress has been achieved in the treatment of non-seminoma tumours of the testis, particularly since cisplatin was introduced. The use of this new drug in combination with other chemotherapeutic agents has resulted in complete remission in over 50% of all advanced cases, and many such remissions that have now persisted for more than two years will probably prove permanent. Thorough analysis of the various prognostic factors involved shows that the probability of response to new chemotherapeutic regimens mainly depends upon the extent of the disease and upon previous treatments. A prospective study of the value of platinum-containing drugs in the treatment of circumscribed tumours is currently under way.

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP.

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide-MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis-Menten kinetics with a maximal velocity V(max) of 2 micromol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.