Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Laparoscopic injury of the obturator nerve during fertility-sparing procedure for cervical cancer

ABSTRACT: BACKGROUND: Intraoperative injury of the obturator nerve has rarely been reported in patients with gynecological malignancies undergoing extensive radical surgeries. Irreversible damage of this nerve causes thigh paresthesia and claudication. Intraoperative repair may be done by end-to-end anastomosis or grafting when achieving tension-free anastomosis is not possible. CASE PRESENTATION: A 28-year-old woman with stage IB cervical cancer underwent fertility--sparing surgery, including conization and bilateral pelvic lymphadenectomy. The left obturator nerve was damaged intraoperatively during pelvic dissection. CONCLUSION: Immediate laparoscopic repair was successful and there was no functional deficit in the left thigh for six months postoperatively.     

Thermal aggregation of ribonuclease A. A contribution to the understanding of the role of 3D domain swapping in protein aggregation

By lyophilizing RNase A from 40% acetic acid solutions, two dimeric aggregates, the "minor" and "major" dimers (named here N-dimer and C-dimer, respectively), form by 3D domain swapping at a ratio of 1:4. Trimeric and tetrameric aggregates are also obtained. The two dimers and the higher oligomers also form without a lyophilization step. By keeping RNase A dissolved at a high concentration (generally 200 mg/ml) in various media at temperatures ranging from 23 to 70 degrees C for times varying from a few minutes to 2 h, various oligomers, in particular the two dimeric conformers, formed in quite different amounts, often inverting their relative quantities depending on the more or less severe unfolding conditions. When unfolding mainly concerned the N terminus of the protein, richer in hydrophilic residues, the N-dimer, formed by 3D domain swapping of the N-terminal alpha-helix of each monomer, prevailed over the C-dimer. Under more vigorous denaturing conditions, where also the C terminus of RNase A, richer in hydrophobic amino acids, unfolded, the C-dimer, formed by 3D domain swapping of the C-terminal beta-strand, prevailed over the other, possibly because of the induction to aggregation promoted by the hydrophobic residues present in the C termini of the two monomers.     

Nitric oxide signaling in plant-pathogen interactions

Nitric oxide (NO), first characterized as an endothelium-derived relaxation factor, is involved in diverse cellular processes including neuronal signaling, blood pressure homeostasis, and immune response. Recent studies have also revealed a role for NO as a signaling molecule in plants. As a developmental regulator, NO promotes germination, leaf extension and root growth, and delays leaf senescence and fruit maturation. Moreover, NO acts as a key signal in plant resistance to incompatible pathogens by triggering resistance-associated hypersensitive cell death. In addition, NO activates the expression of several defense genes (e.g. pathogenesis-related genes, phenylalanine ammonialyase, chalcone synthase) and could play a role in pathways leading to systemic acquired resistance.     

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 ??-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.     

Acute myocardial infarction: Circadian, weekly, and seasonal patterns of occurrence

Convincing evidence has demonstrated that cardiovascular diseases do not occur randomly throughout the day, the week, or the year but show a well defined temporal organization. This article will review circadian, weekly, and seasonal patterns of occurrence of acute myocardial infarction, along with their underlying pathophysiological triggering factors.     

Milk-Based Nutraceutical for Treating Autoimmune Arthritis via the Stimulation of IL-10- and TGF-β-producing CD39+ Regulatory T Cells

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39⁺ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.     

DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

RNASEH1 Mutations Impair mtDNA Replication and Cause Adult-Onset Mitochondrial Encephalomyopathy

Chronic progressive external ophthalmoplegia (CPEO) is common in mitochondrial disorders and is frequently associated with multiple mtDNA deletions. The onset is typically in adulthood, and affected subjects can also present with general muscle weakness. The underlying genetic defects comprise autosomal-dominant or recessive mutations in several nuclear genes, most of which play a role in mtDNA replication. Next-generation sequencing led to the identification of compound-heterozygous RNASEH1 mutations in two singleton subjects and a homozygous mutation in four siblings. RNASEH1, encoding ribonuclease H1 (RNase H1), is an endonuclease that is present in both the nucleus and mitochondria and digests the RNA component of RNA-DNA hybrids. Unlike mitochondria, the nucleus harbors a second ribonuclease (RNase H2). All affected individuals first presented with CPEO and exercise intolerance in their twenties, and these were followed by muscle weakness, dysphagia, and spino-cerebellar signs with impaired gait coordination, dysmetria, and dysarthria. Ragged-red and cytochrome c oxidase (COX)-negative fibers, together with impaired activity of various mitochondrial respiratory chain complexes, were observed in muscle biopsies of affected subjects. Western blot analysis showed the virtual absence of RNase H1 in total lysate from mutant fibroblasts. By an in vitro assay, we demonstrated that altered RNase H1 has a reduced capability to remove the RNA from RNA-DNA hybrids, confirming their pathogenic role. Given that an increasing amount of evidence indicates the presence of RNA primers during mtDNA replication, this result might also explain the accumulation of mtDNA deletions and underscores the importance of RNase H1 for mtDNA maintenance.     

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.