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Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.

Invasion of human erythrocytes is essential for Plasmodium falciparum parasite survival and pathogenesis, and is also a complex phenotype. While some later steps in invasion appear to be invariant and essential, the earlier steps of recognition are controlled by a series of redundant, and only partially understood, receptor-ligand interactions. Reverse genetic analysis of laboratory adapted strains has identified multiple genes that when deleted can alter invasion, but how the relative contributions of each gene translate to the phenotypes of clinical isolates is far from clear. We used a forward genetic approach to identify genes responsible for variable erythrocyte invasion by phenotyping the parents and progeny of previously generated experimental genetic crosses. Linkage analysis using whole genome sequencing data revealed a single major locus was responsible for the majority of phenotypic variation in two invasion pathways. This locus contained the PfRh2a and PfRh2b genes, members of one of the major invasion ligand gene families, but not widely thought to play such a prominent role in specifying invasion phenotypes. Variation in invasion pathways was linked to significant differences in PfRh2a and PfRh2b expression between parasite lines, and their role in specifying alternative invasion was confirmed by CRISPR-Cas9-mediated genome editing. Expansion of the analysis to a large set of clinical P. falciparum isolates revealed common deletions, suggesting that variation at this locus is a major cause of invasion phenotypic variation in the endemic setting. This work has implications for blood-stage vaccine development and will help inform the design and location of future large-scale studies of invasion in clinical isolates., Author summary Plasmodium parasites cause more than 200 million cases of malaria each year. All the symptoms of malaria are caused after Plasmodium parasites invade human red blood cells. Once inside, they grow, multiply and break open the red blood cells to release new parasites. This cycle is repeated every 48 hours, rapidly amplifying the number of parasites and causing severe anemia and other complications. Plasmodium falciparum, the parasite species responsible for almost all malaria deaths, can use multiple different pathways to invade human red blood cells, but the relative importance of each is not well understood. We tested the invasion pathways used by a collection of closely related parasites and compared their genome sequences to identify the genes responsible. This analysis revealed that expression differences in two neighboring genes of the Reticulocyte Binding Homologue family are responsible for most of the variation in two invasion pathways. P. falciparum may use variation in these genes to avoid the immune system or adapt to specific blood groups, which has important implications for vaccine development against malaria.

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3′ splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a neurodevelopmental disorder characterized by epileptic seizures, severe intellectual disability, and autistic features. Mice lacking CDKL5 display multiple behavioral abnormalities reminiscent of the disorder, but the cellular origins of these phenotypes remain unclear. Here, we find that ablating CDKL5 expression specifically from forebrain glutamatergic neurons impairs hippocampal-dependent memory in male conditional knock-out mice. Hippocampal pyramidal neurons lacking CDKL5 show decreased dendritic complexity but a trend toward increased spine density. This morphological change is accompanied by an increase in the frequency of spontaneous miniature EPSCs and interestingly, miniature IPSCs. Using voltage-sensitive dye imaging to interrogate the evoked response of the CA1 microcircuit, we find that CA1 pyramidal neurons lacking CDKL5 show hyperexcitability in their dendritic domain that is constrained by elevated inhibition in a spatially and temporally distinct manner. These results suggest a novel role for CDKL5 in the regulation of synaptic function and uncover an intriguing microcircuit mechanism underlying impaired learning and memory.SIGNIFICANCE STATEMENT Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although Cdkl5 constitutive knock-out mice have recapitulated key aspects of human symptomatology, the cellular origins of CDKL5 deficiency-related phenotypes are unknown. Here, using conditional knock-out mice, we show that hippocampal-dependent learning and memory deficits in CDKL5 deficiency have origins in glutamatergic neurons of the forebrain and that loss of CDKL5 results in the enhancement of synaptic transmission and disruptions in neural circuit dynamics in a spatially and temporally specific manner. Our findings demonstrate that CDKL5 is an important regulator of synaptic function in glutamatergic neurons and serves a critical role in learning and memory.

Fragile X syndrome is a genetic condition resulting fromFMR1gene mutation that leads to intellectual disability, autism-like symptoms, and sensory hypersensitivity. Arbaclofen, a GABA-B agonist, has shown efficacy in some individuals with FXS but has become unavailable after unsuccessful clinical trials, prompting interest in publicly available, racemic baclofen. The present study investigated whether racemic baclofen can remediate abnormalities of neural circuit function, sensory processing, and behavior inFmr1knockout mice, a rodent model of fragile X syndrome.Fmr1knockout mice showed increased baseline and auditory-evoked high-frequency gamma (30–80 Hz) power relative to C57BL/6 controls, as measured by electroencephalography. These deficits were accompanied by decreased T maze spontaneous alternation, decreased social interactions, and increased open field center time, suggestive of diminished working memory, sociability, and anxiety-like behavior, respectively. Abnormal auditory-evoked gamma oscillations, working memory, and anxiety-related behavior were normalized by treatment with baclofen, but impaired sociability was not. Improvements in working memory were evident predominantly in mice whose auditory-evoked gamma oscillations were dampened by baclofen. These findings suggest that racemic baclofen may be useful for targeting sensory and cognitive disturbances in fragile X syndrome.

Molecular biology is a rapidly evolving field that has led to the development of increasingly sophisticated technologies to improve our capacity to study cellular processes in much finer detail. Transcription is the first step in protein expression and the major point of regulation of the components that determine the characteristics, fate and functions of cells. The study of transcriptional regulation has been greatly facilitated by the development of reporter genes and transcription factor expression vectors, which have become versatile tools for manipulating promoters, as well as transcription factors in order to examine their function. The understanding of promoter complexity and transcription factor structure offers an insight into the mechanisms of transcriptional control and their impact on cell behaviour. This review focuses on some of the many applications of molecular cut-and-paste tools for the manipulation of promoters and transcription factors leading to the understanding of crucial aspects of transcriptional regulation.

The Oct transcription factors recognise an octamer DNA element from which they regulate transcription of specific target genes. Oct-1 is the only member of the subfamily that is ubiquitously expressed and has a wide role in transcriptional control. Through interaction with various partner proteins, Oct-1 can modulate accessibility to the chromatin to recruit the transcription machinery and form the pre-initiation complex. The recruited PolII is induced to initiate transcription and stalled until elongation is triggered on interaction with signalling transcription factors. In this way, Oct-1 can fulfil general roles in transcription by opening the chromatin as well as transduce extracellular signals by relaying activation through various interacting partners. The emerging picture of Oct-1 is that of a complex and versatile transcription factor with fundamental functions in cell homeostasis and signal response in general as well as cell specific contexts. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin.

The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types.

Considering the confusing biodistribution data through the literature and few reported alerts as well as our preliminary biodistribution results, we decided to evaluate the interaction and interference of the commonly present (99m) Tc (technetium-99m)-stannic oxide colloid during the direct stannous chloride (99m) Tc-labeling procedure and to assess its influence on the biodistribution pattern of amphiphilic poly(lactic-co-glycolic acid) nanoparticles. In order to confirm our thesis, beside stannous chloride, we employed two different reducing agents that don't form colloidal particles. The use of sodium borohydride was previously reported in the literature, whereas sodium dithionite was adapted for the first time in the (99m) Tc direct labeling procedure for nanoparticles. The results in our paper clearly differentiate among samples with and without colloidal impurities originating from the labeling procedure with a logical follow up of the radiochemical, physicochemical evaluation, and biodistribution studies clarifying previously reported data on stannic oxide colloidal interference. (99m) Tc-nanoparticle complex labeled with sodium dithionite as reducing agent illustrated appropriate labeling efficacy, stability, and potential for further use in biodistribution studies thus providing solution for the problem of low-complex stability when sodium borohydride is used and colloidal stannic oxide interference for stannous chloride procedure.

Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.

The rat PC12 variant cell line, A35C, lacks regulated secretory organelles due to a selective transcriptional block. Hence, A35C may provide clues about the mechanisms that underlie control of neurosecretion. We used mRNA microarray profiling to examine gene expression in A35C. Genes for regulated secretory proteins were down-regulated, while other membrane trafficking pathways were unaffected. A subset of genes repressed in A35C contain binding sites for the neuronal transcriptional repressor, RE1-silencing transcription factor (REST), and REST is expressed in A35C but not normal PC12 cells. Blocking the activity of REST in A35C using a dominant-negative construct induced the reappearance of mRNAs for synaptophysin, chromogranin A, synaptotagmin IV and the beta3 subunit of the voltage-gated sodium channel (Scn3b), all of which contain RE1 sites in their genes. In the case of Scn3b, the corresponding protein was also re-expressed. Granule and synaptic vesicle proteins were not re-expressed at the protein level, despite reactivation of their mRNA, suggesting the existence of additional post-transcriptional control for these proteins. Our work identifies one of the mechanisms underlying the phenotype of neurosecretory-deficient neuroendocrine cells, and begins to define the critical components that determine a key aspect of the neuroendocrine phenotype.

Chromogranin A (CgA) may be critical for secretory granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of secretory granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The secretory granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense granules approximately 180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated secretory pathway and thereby rescue the sorting of other secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of secretory granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.

Electrical excitability in neurons depends on the expression and activity of voltage-gated sodium channels in the neuronal plasma membrane. The ion-conducting alpha-subunit of the channel is associated with auxiliary beta-subunits of which there are four known types. In the present study, we describe the first detailed structure/function analysis of the beta3-subunit. We correlate the effect of point mutations and deletions in beta3 with the functional properties of the sodium channel and its membrane-targeting behaviour. We show that the extracellular domain influences sodium channel gating properties, but is not required for the delivery of beta3 to the plasma membrane when expressed with the alpha-subunit. In contrast, the intracellular domain is essential for correct subunit targeting. Our results reveal the crucial importance of the Cys21-Cys96 disulphide bond in maintaining the functionally correct beta3 structure and establish a role for a second putative disulphide bond (Cys2-Cys24) in modulating channel inactivation kinetics. Surprisingly, our results imply that the wild-type beta3 molecule can traverse the secretory pathway independently of the alpha-subunit.

Nitric oxide (NO) produced by inducible nitric-oxide synthase (NOSII) is mainly regulated at the transcriptional level by the nuclear factor-kappaB (NF-kappaB). In the present study, we further analyzed the role of NF-kappaB in the in vivo transcriptional regulation of NOSII gene by comparing two clones isolated from the EMT-6 mouse mammary cancer cell line. In response to interleukin (IL)-1beta or lipopolysaccharide (LPS), EMT-6 clone J (EMT-6J) cells produce 3-fold more NO than EMT-6 clone H (EMT-6H) cells, an effect correlated with enhanced activation of NF-kappaB in EMT-6J cells. In response to IL-1beta, the kinetics of degradation of NF-kappaB inhibitors IkappaB-alpha and IkappaB-beta, the nucleo-cytoplasmic shuttling of the transcription factor and its binding to a specific DNA sequence were similar in both clones. In contrast, an IL-1beta-induced phosphorylation of serine residues in NF-kappaB p65 subunit was observed in EMT-6J, but not in EMT-6H, cells. This IL-1beta-induced phosphorylation of p65 was specifically prevented by pretreatment of EMT-6J cells with the casein kinase II inhibitor DRB. Small interfering RNA-mediated depletion of casein kinase II-alpha subunit also decreased NF-kappaB transcriptional activity and NOSII gene transcription in IL-1beta and LPS-stimulated EMT-6J cells to the levels observed in EMT-6H cells treated in the same conditions. Altogether, these data indicate that casein kinase II-mediated phosphorylation of p65 subunit can enhance the transcriptional activity of NF-kappaB in vivo. This post-translational modification of the transcription factor can be responsible for increased NOSII gene transcription and NO production in tumor cells exposed to either IL-1beta or LPS.

Despite the wide range of available therapies, human colon cancers remain difficult to cure. Evidence for efficient antitumoral immune responses to be raised is now widely accepted, and numerous strategies exploiting the host immune system have been developed. A treatment based on the lipid-A derivative OM-174 has been developed in our laboratory. OM-174 induces the rejection of tumors established by injection of PROb colon cancer cells in syngeneic BDIX rats. Our immunohistochemistry study demonstrated that OM-174 treatment is associated with tumor cell apoptosis. Caspase 3 activation was detected 24 h after the first OM-174 injection. Six days after the beginning of the treatment, dendritic cells were the first immune cells that invaded tumor nodules. When dendritic cells came into contact with apoptotic tumor cells, an increased expression of the costimulatory molecules B7-1 and B7-2 was detected at the surface of these cells. Five days later, macrophages were found in the tumor nodules. Lymphocytes organized into a crown surrounding the nodules that progressively regressed during the treatment. T lymphocytes were not in contact with tumor cells or apoptotic cells at any time point. The kinetics of tumor cell apoptosis induced by OM-174, as well as the appearance of innate followed by adaptative immune cells in the tumor nodules, were compatible with cell activation and the development of immune response.

HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-alpha). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-kappaB by degrading its main inhibitor, I-kappaBalpha. HSP27 overexpression increases NF-kappaB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and interleukin 1beta. HSP27 does not affect I-kappaBalpha phosphorylation but enhances the degradation of phosphorylated I-kappaBalpha by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-kappaBalpha. A protein complex that includes HSP27, phosphorylated I-kappaBalpha, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-kappaBalpha. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-kappaB activity.

Cancer is the second cause of death in developed countries. Surgery and radiotherapy are widely used in the treatment of localized and attainable tumors. Chemotherapy and immunotherapy are used as adjuvant treatments or as therapies for advanced-stage diseases. However, chemotherapy often leads to a high relapse rate because of drug resistance, which might be overcome by immunotherapy. When the immune system is not destroyed by chemotherapy, it is able to recognize tumor-specific antigens and eventually can eliminate the cancer cells. One of the means of achieving this goal is the use of lipopolysaccharides (LPSs). LPSs are components of the outer membrane of Gram-negative bacteria and are composed of a polysaccharide, an oligosaccharide core, and lipid A. These compounds have the property of inducing the secretion of various cytokines, such as tumor necrosis factor-α (TNF-α),1interferon-γ (IFN-γ),2interleukin 1-β (IL1-β),3and interleukin-6 (IL-6),4as well as activating immune cells, including neutrophils,5macrophages,6and CD4 and CD8 T lymphocytes that infiltrate the tumors.7,8Furthermore, these compounds have been shown to decrease suppressive cytokines such as transforming growth factor-β (TGF-β).4,9 The first assays with bacterial extracts containing LPS to be used as a treatment for cancer were performed in clinical trials in 1898.10A half-century later, the antitumoral effect was attributed to LPSs in murine subcutaneous tumors,11and eventually it was demonstrated that this effect is due to the lipid A component of LPSs.12Because lipid A and its derivatives are less toxic than LPSs, most anticancer treatments aimed at activating an immune response against tumors have been developed with the use of natural lipid A or synthetic analogs, either alone or as adjuvants, to enhance the efficacy of therapeutic anticancer vaccines. Animal models permit the investigation of the mechanisms of the …

Bacterial lipopolysaccharide (LPS) and its active component, lipid A, have been used either alone or as adjuvant in therapeutic anticancer vaccines. Lipid A induces various transcription factors via intracellular signaling cascades initiated by their receptor CD14-TLR4. These events lead to the synthesis of cytokines, which either have direct cytotoxic effect or stimulate the immune system. Their antitumoral effect has been demonstrated in animal models as well as clinical trials. Studies in animal models showed that their antitumoral effect relies mostly on the generation of an effective immune response. In humans, the antitumoral effect was correlated with an antibody response and cell-mediated cytotoxicity. So far, some encouraging results have been achieved in phase I and II clinical trials with regards to response and stabilization of the disease, but an expansion of the studies and trials is needed to find the best conditions for their clinical application.

There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis. Using in vivo footprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ?898/9, at a putative partial heat shock element (HSE) and at -893/4, a site bordering an E-box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions. LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone. Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E-box and a partial HSE within the iNOS enhancer. Binding to the E-box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments. The proteins occupying this site were identified as belonging to the USF family of transcription factors. Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heeat shock factor-1 (HSF-1). Mutations at the HSE revealed the importance of HSF-1 in the induction of iNOS by LPS. Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene. These results could have implications in the host response mechanism to fever-associated gram-negative infection.

A variant of the PC12 pheochromocytoma cell line (termed A35C) has been isolated that lacks regulated secretory organelles and several constituent proteins. Northern and Southern blot analyses suggested a block at the transcriptional level. The proprotein-converting enzyme carboxypeptidase H was synthesised in the A35C cell line but was secreted by the constitutive pathway. Transient transfection of A35C cells with cDNAs encoding the regulated secretory proteins dopamine beta-hydroxylase and synaptotagmin I resulted in distinct patterns of mistargeting of these proteins. It is surprising that hybrid cells created by fusing normal PC12 cells with A35C cells exhibited the variant phenotype, suggesting that A35C cells express an inhibitory factor that represses neuroendocrine-specific gene expression.

The proteolytic pattern of Trypanosoma cruzi epimastigotes was studied using electrophoresis in gelatin containing SDS polyacrylamide gels. Under conditions that represent a nutritional stress such as the depletion of glucose in an axenic medium the results obtained showed a significant increase in the activity of two proteases of M(r) = 50,000 and M(r) = 60,000 as well as the appearance of higher molecular weight bands during the transition from log to stationary phase parasites. The use of different protease inhibitors strongly suggests that all these proteases are of the cysteine type, and their acid pH optimal would be in agreement with the possible localization in the parasite lysosomes. The reincubation of stationary phase parasites in fresh LIT medium or the addition of 0.4% glucose partially reverted the proteolytic pattern to that observed in log phase parasites. The regulation in the activity of these enzymes could be related to the well established use of amino acids for catabolic purpose after depletion of the medium glucose.

Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.

Proteolysis of endogenous proteins may play a key role in the adaptation of T. cruzi to the different host environments to which it is exposed during its complex life cycle. For this reason, we have attempted to study the intracellular pathways of protein degradation in the non infective epimastigotes form (EP strain) of T. cruzi. Following intracellular proteolysis by pulse chase experiments with 35 S methionine, we observed a significant inhibition (50%) of the degradation of endogenous proteins in log phase parasites in the presence of inhibitors of lysosomal functions, such as chloroquine and E 64. A significant increase in proteolysis was observed in stationary phase parasites which was reverted to log phase values by supplementing the chase medium with 0.5% glucose or 10% serum, or in the presence of chloroquine. Under this condition of nutritional stress, we could observe an increase in the activity of acid proteases. A significant increase in the degradation rates was observed when abnormal proteins were induced in the parasite by amino acid analogs and puromycin. This increase was not affected by E 64, suggesting the participation of non lysosomal mechanisms in the degradation of rapidly degradable abnormal proteins. Under these conditions, we could observe an increase in high molecular weight conjugates of ubiquitin with respect to endogenous proteins. These results suggest the importance of lysosomal mechanisms in the degradation of cellular proteins in nutritional optimal conditions and during nutritional deprivation, and the possible involvement of the ubiquitin system in the degradation of high turnover proteins.

Heat shock protein 70 (HSP70) can inhibit apoptosis by neutralizing and interacting with apoptosis-inducing factor (AIF), a mitochondrial flavoprotein that translocates upon apoptosis induction to the nucleus, via the cytosol. Here, we show that only members of the HSP70 family interact with AIF. Systematic deletion mapping revealed the existence of three distinct functional regions in the AIF protein: (1) a region between amino acids 150 and 228 that binds HSP70, (2) a domain between residues 367 and 459 that includes a nuclear localization sequence (NLS) and (3) a C-terminal domain beyond residue 567 required for its chromatin-condensing activity. Deletion of the 150-268 domain completely abolished HSP70 binding and facilitated the nuclear import of AIF, resulting in a gain-of-function phenotype with enhanced AIF-mediated chromatin condensation as compared to wild-type AIF. This gain-of-function phenotype was observed in wild-type control cells (which express low but significant levels of HSP70), yet was lost when AIFDelta150-268 was introduced into HSP70 knockout cells, underscoring the functional importance of the AIF-HSP70 interaction. Altogether, our data demonstrate that AIF inhibition by HSP70 involves cytosolic retention of AIF. Moreover, it appears that endogenous HSP70 protein levels are sufficiently elevated to modulate the lethal action of AIF.