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A 22-week pregnancy was terminated after discovery of serious echographic abnormalities. Fetal examination showed cyclopia, sacral meningocele, and syndactyly. The karyotype was 69,XXX. The parents had identical HLA alleles A1, A2, and Bw21. The mechanism of the triploidy was determined by chromosome marker analysis to be digyny. The association of triploidy with holoprosencephaly and the parents' identical immunological status are discussed. [ABSTRACT FROM PUBLISHER]

The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKβ and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBβ) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/β dimerization in substrate motif recognition., (© 2024. The Author(s).)

Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying V H Hs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the V H Hs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Chromobodies made of nanobodies fused to fluorescent proteins are powerful tools for targeting and tracing intracellular proteins in living cells. Typically, this is achieved by transfecting plasmids encoding the chromobodies. However, an excess of unbound chromobody relative to the endogenous antigen can result in high background fluorescence in live cell imaging. Here, we overcome this problem by using mRNA encoding chromobodies. Our approach allows one to precisely control the amount of chromobody expressed inside the cell by adjusting the amount of transfected mRNA. To challenge our method, we evaluate three chromobodies targeting intracellular proteins of different abundance and cellular localization, namely lamin A/C, Dnmt1 and actin. We demonstrate that the expression of chromobodies in living cells by transfection of tuned amounts of the corresponding mRNAs allows the accurate tracking of their cellular targets by time-lapse fluorescence microscopy., (© 2024 Wiley-VCH GmbH.)

Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed V H Hs and the antigen present in the cell, preventing the necessity to remove the excess unbound V H Hs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system.

Advances in cryo-electron microscopy (EM) enable imaging of protein assemblies within mammalian cells in a near native state when samples are preserved by cryogenic vitrification. To accompany this progress, specialized EM labelling protocols must be developed. Gold nanoparticles (AuNPs) of 2 nm are synthesized and functionalized to bind selected intracellular targets inside living human cells and to be detected in vitreous sections. As a proof of concept, thioaminobenzoate-, thionitrobenzoate-coordinated gold nanoparticles are functionalized on their surface with SV40 Nuclear Localization Signal (NLS)-containing peptides and 2 kDa polyethyleneglycols (PEG) by thiolate exchange to target the importin-mediated nuclear machinery and facilitate cytosolic diffusion by shielding the AuNP surface from non-specific binding to cell components, respectively. After delivery by electroporation into the cytoplasm of living human cells, the PEG-coated AuNPs diffuse freely in the cytoplasm but do not enter the nucleus. Incorporation of NLS within the PEG coverage promotes a quick nuclear import of the nanoparticles in relation to the density of NLS onto the AuNPs. Cryo-EM of vitreous cell sections demonstrate the presence of 2 nm AuNPs as single entities in the nucleus. Biofunctionalized AuNPs combined with live-cell electroporation procedures are thus potent labeling tools for the identification of macromolecules in cellular cryo-EM., (© 2023 The Authors. Small Methods published by Wiley-VCH GmbH.)

The homogeneous labeling of antibodies and their fragments is a critical step for the generation of robust probes used in immuno-detection applications. To date, numerous chemical, genetic and peptide-based site-specific coupling methods have been developed. Among these methods, co-assembling peptide-tags is one of the most straightforward and versatile solutions. Here, we describe site-specific labeling of nanobodies through the use of two co-associating peptides tags, E3 and K3, originating from the tetramerization domain of p53. These E3 and K3-tags provide a simple and robust method for associating stoichiometric amount of V H H and fluorescent probes, either fluorescent proteins or fluorochromes, at specific positions. As a proof of concept, a nanobody targeting the human epidermal growth factor receptor 2 (HER2), the nano-HER2 was genetically fused to the E3 and associated with different fluorescent K3-derivates. Entities were produced separately in Escherichia coli in soluble forms at high yields and co-assembled in vitro. These molecular probes present high binding specificity on HER2-overexpressing cells in flow-cytometry with relative binding constants in the low nanomolar range and are stable enough to stain HER2-receptor on living cells followed detection using fluorescent confocal microscopy. Altogether, our results demonstrate that the non-covalent conjugation method using these two co-associating peptides can be easily implemented for the modular engineering of molecular probes for cell immuno-staining.

Bivalent V H Hs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both V H Hs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Advances in microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders originating from the immune response arsenal to gold nanoparticles (AuNPs) as contrasting agents is the method of choice to obtain labeling tools. However, conjugation of the minimal sized 15 kDa nanobody (Nb) to AuNPs remains challenging in comparison to the conjugation of 150 kDa IgG to AuNPs. Herein, effective Nb-AuNP assemblies are built using the selective and almost irreversible non-covalent associations between two peptide sequences deriving from a p53 heterotetramer domain variant. The 15 kDa GFP-binding Nb is fused to one dimerizing motif to obtain a recombinant Nb dimer with improved avidity for GFP while the other complementing dimerizing motif is equipped with thiols and grafted to a 2.4 nm substituted thiobenzoate-coordinated AuNP via thiolate exchange. After pegylation, the modified AuNPs are able to non-covalently anchor Nb dimers and the subsequent complexes demonstrate the ability to form immunogold label GFP-protein fusions within various subcellular locations. These tools open an avenue for precise localization of targets at high resolution by electron microscopy., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC 50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

Monitoring the assembly of macromolecules to design entities with novel properties can be achieved either chemically creating covalent bonds or by noncovalent connections using appropriate structural motifs. In this report, two self-associating peptides (named K3 and E3) that originate from p53 tetramerization domain were developed as tools for highly specific and noncovalent heterotetramerization of two biomolecules. The pairing/coupling preferences of K3 and E3 were first evaluated by molecular modeling data and confirmed using circular dichroism spectroscopy, size-exclusion chromatography, and biological assays. Regardless of the moieties fused to K3 and E3, these two peptides self-assembled into dimers of dimers to form bivalent heterotetrameric complexes that proved to be extremely stable inside living cells. The benefits of the multivalency in terms of avidity, specificity, and expanded functional activity were strikingly revealed when the proliferating cell nuclear antigen (PCNA), which is essential for DNA replication, was targeted using a heterotetramer presenting both an antibody fragment against PCNA and a specific PCNA binder peptide. In vitro heterotetramerization of these two known PCNA ligands increased their binding efficiencies to PCNA up to 80-fold compared to the best homotetramer counterpart. In cellulo, the heterotetramers were able to efficiently inhibit DNA replication and to trigger cell death. Altogether, we demonstrate that these two biselective self-assembling peptidic domains offer a versatile noncovalent conjugation method that can be easily implemented for protein engineering.

Therapeutic monoclonal antibodies benefit to patients and the conjugation to gold nanoparticles (AuNPs) might bring additional activities to these macromolecules. However, the behavior of the conjugate will largely depend on the bulkiness of the AuNP and small sizes are moreover preferable for diffusion. Water-soluble thiolate-protected AuNPs having diameters of 2-3 nm can be synthesized with narrow polydispersity and can selectively react with incoming organic thiols via a S N 2-like mechanism. We therefore synthesized a mixed thionitrobenzoic acid- , thioaminobenzoic acid-monolayered AuNP of 2.4 nm in diameter and developed a site-selective conjugation strategy to link the AuNP to Cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody used in clinic. The water-soluble 80 kDa AuNP was fully characterized and then reacted to the hinge area of Cetuximab, which was selectively reduced using mild concentration of TCEP. The conjugation proceeded smoothly and could be analyzed by polyacrylamide gel electrophoresis, indicating the formation of a 1:1 AuNP-IgG conjugate as the main product. When added to EGFR expressing glioblastoma cells, the AuNP-Cetuximab conjugate selectively bound to the cell surface receptor, inhibited EGFR autophosphorylation and entered into endosomes like Cetuximab. Altogether, we describe a simple and robust protocol for a site-directed conjugation of a thiolate-protected AuNP to Cetuximab, which could be easily monitored, thereby allowing to assess the quality of the product formation. The conjugated 2.4 nm AuNP did not majorly affect the biological behavior of Cetuximab, but provided it with the electronic properties of the AuNP. This offers the ability to detect the tagged antibody and opens application for targeted cancer radiotherapy.

Here we developed a complementation method for the study of essential genes in live human cells using the CRISPR/Cas9 system. Proteins encoded by essential genes were expressed using a derivative of the pCEP4 compensating plasmid in combination with Cas9 endonuclease targeting of the chromosomal genes. We show that this strategy can be applied to essential genes, such as those coding for proliferating cell nuclear antigen ( PCNA ) and DNA polymerase delta subunit 2 ( POLD2 ). As demonstrated for the PCNA protein, our method allows mutational analysis of essential protein-coding sequences in live cells.

The transcription factor ATF7 undergoes multiple post-translational modifications, each of which has distinct effects upon ATF7 function. Here, we show that ATF7 phosphorylation on residue Thr112 exclusively occurs during mitosis, and that ATF7 is excluded from the condensed chromatin. Both processes are CDK1/cyclin B dependent. Using a transduced neutralizing monoclonal antibody directed against the Thr112 epitope in living cells, we could demonstrate that Thr112 phosphorylation protects endogenous ATF7 protein from degradation, while it has no effect on the displacement of ATF7 from the condensed chromatin. The crucial role of Thr112 phosphorylation in stabilizing ATF7 protein during mitosis was confirmed using phospho-mimetic and phospho-deficient mutants. Finally, silencing ATF7 by CRISPR/Cas9 technology leads to a decrease of cyclin D1 protein expression levels. We propose that mitotic stabilized ATF7 protein re-localizes onto chromatin at the end of telophase and contributes to induce the cyclin D1 gene expression.

Background: Multiple myeloma is characterized by clonal expansion of B cells producing monoclonal immunoglobulins or fragments thereof, which can be detected in the serum and/or urine and are ideal target antigens for patient-specific immunotherapies., Methods: Using phage particles as immunological carriers, we employed a novel chemically linked idiotype vaccine in a clinical phase I/II trial including 15 patients with advanced multiple myeloma. Vaccines composed of purified paraproteins linked to phage were manufactured successfully for each patient. Patients received six intradermal immunizations with phage idiotype vaccines in three different dose groups., Results: Phage idiotype was well tolerated by all study participants. A subset of patients (80% in the middle dose group) displayed a clinical response indicated by decrease or stabilization of paraprotein levels. Patients exhibiting a clinical response to phage vaccines also raised idiotype-specific immunoglobulins. Induction of a cellular immune response was demonstrated by a cytotoxicity assay and delayed type hypersensitivity tests., Conclusion: We present a simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible patient-specific therapy for patients with advanced multiple myeloma and produced promising anti-tumor activity in a subset of patients.

Background: B cell malignancies are characterized by clonal expansion of B cells expressing tumor-specific idiotypes on their surface. These idiotypes are ideal target antigens for an individualized immunotherapy. However, previous idiotype vaccines mostly lacked efficiency due to a low immunogenicity of the idiotype. The objective of the present study was the determination of the feasibility, safety and immunogenicity of a novel chemically linked phage idiotype vaccine., Methods: In the murine B cell lymphoma 1 model, tumor idiotypes were chemically linked to phage particles used as immunological carriers. For comparison, the idiotype was genetically expressed on the major phage coat protein g8 or linked to keyhole limpet hemocynanin. After intradermal immunizations with idiotype vaccines, tolerability and humoral immune responses were assessed., Results: Feasibility and tolerability of the chemically linked phage idiotype vaccine was demonstrated. Vaccination with B cell lymphoma 1 idiotype expressing phage resulted in a significant survival benefit in the murine B cell lymphoma 1 protection model (60.2±23.8 days vs. 41.8±1.6 days and 39.8±3.8 days after vaccination with wild type phage or phosphate buffered saline, respectively). Superior immunogenicity of the chemically linked phage idiotype vaccine compared to the genetically engineered phage idiotype and keyhole limpet hemocynanin-coupled idiotype vaccine was demonstrated by significantly higher B cell lymphoma 1 idiotype-specific IgG levels after vaccination with chemically linked phage idiotype., Conclusion: We present a novel, simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible therapy and may produce a superior immune response compared to previously employed idiotype vaccination strategies.

Objectives: Analgesic protocols administered before a follicular puncture under local anesthesia are well tolerated when using NSAIDs, but we still do not know their possible impacts on in vitro fertilization (IVF) outcomes., Material and Methods: A retrospective monocentric study using two consecutive temporal cohorts of patients was conducted to compare two analgesic protocols: paracetamol/alprazolam (P/A), then nefopam/ketoprofen (N/K)., Results: We demonstrated that biochemical pregnancy rate and the others outcomes of IVF are not significantly influenced by the type of analgesic protocol used., Conclusion: The protocol N/K enhances patient comfort without jeopardizing the IVF success rates., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Alternative splicing and post-translational modifications are processes that give rise to the complexity of the proteome. The nuclear ATF7 and ATF2 (activating transcription factor) are structurally homologous leucine zipper transcription factors encoded by distinct genes. Stress and growth factors activate ATF2 and ATF7 mainly via sequential phosphorylation of two conserved threonine residues in their activation domain. Distinct protein kinases, among which mitogen-activated protein kinases (MAPK), phosphorylate ATF2 and ATF7 first on Thr71/Thr53 and next on Thr69/Thr51 residues respectively, resulting in transcriptional activation. Here, we identify and characterize a cytoplasmic alternatively spliced isoform of ATF7. This variant, named ATF7-4, inhibits both ATF2 and ATF7 transcriptional activities by impairing the first phosphorylation event on Thr71/Thr53 residues. ATF7-4 indeed sequesters the Thr53-phosphorylating kinase in the cytoplasm. Upon stimulus-induced phosphorylation, ATF7-4 is poly-ubiquitinated and degraded, enabling the release of the kinase and ATF7/ATF2 activation. Our data therefore conclusively establish that ATF7-4 is an important cytoplasmic negative regulator of ATF7 and ATF2 transcription factors.

The ubiquitous activating transcription factor (ATF) 7 binds as a homodimer to the cAMP response element/TPA response element motifs present in the promoters of its target genes. ATF7 is homologous to ATF2 and heterodimerizes with Jun or Fos proteins, modulating their DNA-binding specificities. We previously demonstrated that TAF12, a component of the TFIID general transcription factor, mediates ATF7 transcriptional activity through direct interactions between the two proteins. By contrast, ATF7, but not ATF2, is modified in vivo by sumoylation, which restricts its subcellular localization, thereby inhibiting its transcriptional activity. In the present study, we dissect the mechanism of this functional switch. We characterized the multisite phosphorylation of the ATF7 activation domain and identified one of the involved kinase, p38beta2 mitogen-activated protein kinase. In addition, we show that epidermal growth factor treatment results in a two-step modification mechanism of ATF7 activation domain. The Thr53 residue is phosphorylated first by a presently unknown kinase, allowing p38beta2 mitogen-activated protein kinase to modify the Thr51 residue, excluding the sumoylation of ATF7 protein. The resulting activation of transcription is related to an increased association of TAF12 with this phosphorylated form of ATF7. Our data therefore conclusively establish that sumoylation and phosphorylation of ATF7 are two antagonistic posttranslational modifications.

Recombinant antibody technology is a rapidly evolving field that enables the study and improvement of antibody properties by means of genetic engineering. Moreover, the functional expression of antibody fragments in Escherichia coli has formed the basis for antibody library generation and selection, a powerful method to produce human antibodies for therapy. Because in vitro-generated antibodies offer various advantages over traditionally produced monoclonal antibodies, such molecules are now increasingly used for standard immunological assays. This chapter will give a short review on how recombinant antibodies are generally be produced and engineered, and how typical immunoassays are performed.

Endotoxins are frequent contaminants of recombinant proteins produced in Escherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg(-1) is, however, one of the most problematic steps during protein purification from E. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced in E. coli using a non-ionic detergent. Endotoxin content was as low as 5 to 9 EU mg(-1) with a recovery of antibody fragments of over 90%. Non-ionic detergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinant antibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.

Endotoxin is an unwanted by product of recombinant proteins purified from Escherichia coli. The inherent toxicity of endotoxins makes their removal an important step for the proteins' application in several biological assays and for safe parenteral administration. The method described in this paper is a one-step protocol which is effective at removing tightly bound endotoxin from over-expressed tagged proteins in E. coli. We combined affinity chromatography with a non-ionic detergent washing step, to remove most of the endotoxin contaminants from the end product. An endotoxin reduction of less than 4 to 0.2 EU mg(-1) was achieved with protein recovery close to a yield 100%. As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense.

Purpose: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell-based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified. Whereas a number of HLA class I-presented TRP-2-derived epitopes are known, information about HLA class II-presented antigenic ligands recognized by CD4+ T helper (Th) cells is limited., Experimental Design: The search for TRP-2-derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1*0301 ligands in combination with peptide and protein immunizations of HLA-DRB1*0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in human T-cell cultures., Results: This strategy led to the characterization of TRP-2(60-74) as an HLA-DRB1*0301-restricted Th epitope. Importantly, TRP-2(60-74)-reactive human CD4+ Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1*03+ melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-2(60-74)-reactive T cells, suggesting that these T cells were already activated in vivo., Conclusion: Peptide TRP-2(60-74) might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients.

Introduction: Our experience of ICSI began in 1997. This study reviews our four-year experience based on 42 consecutive couples., Material and Method: Between February 1997 and December 2000, 42 sterile couples due to male infertility were treated by ICSI. Surgical exploration and "open" gamete collection were proposed regardless of the predefined type of azoospermia, obstructive or non-obstructive., Result: Seventy one cycles were performed: ICSI used epididymal spermatozoa in 49 cycles, and testicular spermatozoa in 22 cycles. The fertilization rate was 76% for fresh semen and 87% for frozen semen. The fertilization rate was 88% for epididymal spermatozoa and 68% for testicular spermatozoa. 13 pregnancies were obtained (18.3%), 11 babies were born at term including 3 twin pregnancies. No significant difference was observed between fresh and frozen semen, or between obstructive and non-obstructive azoospermia., Conclusion: IVF-ICSI applied to sterility due to male infertility has revolutionized the management of sterile couples. However, as for any new procedure in medicine, we must be vigilant, as a sufficient follow-up is necessary to definitively evaluate the safety of ICSI, especially in terms of the risk of genetic abnormalities.

Protein translocation across the mitochondrial inner membrane is driven by cycles of binding and release of mitochondrial heat shock protein 70 (mtHsp70) in the matrix. The peripheral inner membrane protein, Tim44, recruits mtHsp70 in an ATP-dependent manner to the import sites. We show that DnaK, the closely related Hsp70 of Escherichia coli, when targeted to the matrix of yeast mitochondria, interacts in a specific manner with Tim44. The interaction is, however, not regulated by ATP, and DnaK cannot support protein translocation. We used truncated mtHsp70s and chimeric proteins consisting of segments of mtHsp70 and DnaK to analyze which portions of mtHsp70 bind and functionally interact with Tim44. We show that Tim44 interacts with the beta-stranded core of the peptide binding domain of mtHsp70 and of DnaK. The alpha-helices A and B of the peptide binding domain of mtHsp70 are required to transmit the nucleotide state of the ATPase domain to the peptide binding domain. Tim44, by interacting in this way with the peptide binding domain, is proposed to coordinate the binding of mtHsp70 to the incoming preprotein and the subsequent release of the mtHsp70-preprotein complex from the TIM23 complex, the translocase of the inner membrane.

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.

Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development. Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation. Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen. In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium. These observations are consistent with the following model. In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels. Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium. The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted. This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.

A retrospective (1987-1995), single-center study was conducted to evaluate the IVF success rate in women who were 40 years and over. Controls were randomly selected patients who were 35 years or younger from the same center. The main evaluation criterion was the number of pregnancies initiated in each group and especially the number of full-term deliveries (take-home baby rate). Differences were considered as statistically significant for P < or = 0.05: A total of 194 IVF attempts in women 40 years or over were compared to 209 attempts in the control group. The mean ages of the two groups was 40.9 vs. 29.3 years (P < 0.001). The duration of follicle stimulation was 12.9 vs. 13.1 days (not significant, NS). The number of ampules was 29.6 vs. 29.2 (NS). Serum estradiol levels were 1435.8 vs. 2020.8 pg/ml (P < 0.001). Oocytes: 4.6 vs. 7.3 (P < 0.001). Embryos: 1.7 vs. 2.8 (P < 0.0001). Full-term deliveries: 3.6 vs. 13.4% (P < 0.05). Better oocyte retrieval was achieved (5.3 vs. 3.3; P = 0.001) in the group that was 40 years or over, but there were no differences in the rate of embryo transfer (1.9 vs. 1.3; NS) and full-term deliveries (4.2 vs. 2.9%) in a long protocol compared to a short one. The results of the study are similar to those found in the literature. Indications for standard IVF without oocyte donation should be carefully thought out and couples should be warned of the low success rate.