Transforming the Niche: The Emerging Role of Extracellular Vesicles in Acute Myeloid Leukaemia Progression.

Acute myeloid leukaemia (AML) management remains a significant challenge in oncology due to its low survival rates and high post-treatment relapse rates, mainly attributed to treatment-resistant leukaemic stem cells (LSCs) residing in bone marrow (BM) niches. This review offers an in-depth analysis of AML progression, highlighting the pivotal role of extracellular vesicles (EVs) in the dynamic remodelling of BM niche intercellular communication. We explore recent advancements elucidating the mechanisms through which EVs facilitate complex crosstalk, effectively promoting AML hallmarks and drug resistance. Adopting a temporal view, we chart the evolving landscape of EV-mediated interactions within the AML niche, underscoring the transformative potential of these insights for therapeutic intervention. Furthermore, the review discusses the emerging understanding of endothelial cell subsets' impact across BM niches in shaping AML disease progression, adding another layer of complexity to the disease progression and treatment resistance. We highlight the potential of cutting-edge methodologies, such as organ-on-chip (OoC) and single-EV analysis technologies, to provide unprecedented insights into AML-niche interactions in a human setting. Leveraging accumulated insights into AML EV signalling to reconfigure BM niches and pioneer novel approaches to decipher the EV signalling networks that fuel AML within the human context could revolutionise the development of niche-targeted therapy for leukaemia eradication.

HDAC-6 inhibition ameliorates the early neuropathology in a mouse model of Krabbe disease.

Introduction: In Krabbe disease (KD), mutations in ß-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport. Methods: We tested whether inhibiting the α-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice. Results: Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria. Discussion: Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction.

Are circulating endothelial cells the next target for transcriptome-level pathway analysis in ARDS?

Acute respiratory distress syndrome (ARDS) has had no mortality-improving pharmacological intervention despite 50 years of high-caliber research due to its heterogeneity (Huppert LA, Matthay MA, Ware LB. Semin Respir Crit Care Med 40: 31-39, 2019). For the field to advance, better definitions for ARDS subgroups that more uniformly respond to therapies are needed (Bos LDJ, Scicluna BP, Ong DSY, Cremer O, van der Poll T, Schultz MJ. Am J Respir Crit Care Med 200: 42-50, 2019; Dickson RP, Schultz MJ, T van der P, Schouten LR, Falkowski NR, Luth JE, Sjoding MW, Brown CA, Chanderraj R, Huffnagle GB, Bos LDJ, Biomarker Analysis in Septic ICU Patients (BASIC) Consortium. Am J Respir Crit Care Med 201: 555-563, 2020; Sinha P, Calfee CS. Am J Respir Crit Care Med 200: 4-6, 2019; Calfee CS, Delucchi K, Parsons PE, Thompson BT, Ware LB, Matthay MA, NHLBI ARDS Network. Lancet Respir Med 2: 611-620, 2014; Hendrickson CM, Matthay MA. Pulm Circ 8: 1-12, 2018). A plethora of high-quality clinical research has uncovered the next generation of soluble biomarkers that provide the predictive enrichment necessary for trial recruitment; however, plasma-soluble markers do not specify the damaged organ of origin nor do they provide insight into disease mechanisms. In this perspective, we make the case for querying the transcriptome of circulating endothelial cells (CECs), which when shed from vessels after inflammatory insult, become heralds of site-specific inflammatory damage. We review the application of CEC quantification to multiple disease phenotypes (including myocardial infarction, vasculitides, cancer, and ARDS), in each case supporting the association of CEC number with disease severity. We also argue for the utility of single-cell RNA transcriptomics to the understanding of cell-specific contributions to disease pathophysiology and its potential to uncover novel insight on signals contributing to CEC shedding in ARDS.

Clinical Profile Among Brazilian Mucopolysaccharidosis type II Patients: Subgroup Analysis from the Hunter Outcome Survey

Abstract Mucopolysaccharidosis type II (MPS II) is a rare genetic, multiorgan disease. Little information about the Brazilian context is available to date; thus, this descriptive subgroup analysis was conducted on Brazilian data from the Hunter Outcome Survey (HOS), including clinical characteristics among MPS II patients from Brazil. HOS is a global, multi-center, long-term, observational registry of patients with MPS II (NCT03292887). Variables related to organ system involvement, signs and symptoms, surgical procedures and survival among Brazilian patients were extracted from HOS database. Data from 153 Brazilian patients with MPS II were analyzed. Musculoskeletal (96.6%), abdomen/gastrointestinal (95.2%), neurological (88.7%), pulmonary (86.2%), and ear (81.3%) were the most frequently observed organ/systems involved. Regarding signs and symptoms, the most prevalent symptom was coarse facial features consistent with the disease (94.6%), followed by joint stiffness and limited function (89.3%), hernia (84.2%) and hepatomegaly (82.2%). Median survival time was 22.0 years, and the major cause of death was respiratory failure (31.8%). These data may be helpful to understand disease characteristics and to help improve the quality of MPS II patient care in Brazil.

Osteoclast-derived extracellular vesicles are implicated in sensory neurons sprouting through the activation of epidermal growth factor signaling.

BACKGROUND: Different pathologies, affecting the skeletal system, were reported to display altered bone and/or cartilage innervation profiles leading to the deregulation of the tissue homeostasis. The patterning of peripheral innervation is achieved through the tissue-specific expression of attractive or repulsive axonal guidance cues in specific space and time frames. During the last decade, emerging findings attributed to the extracellular vesicles (EV) trading a central role in peripheral tissue innervation. However, to date, the contribution of EV in controlling bone innervation is totally unknown. RESULTS: Here we show that sensory neurons outgrowth induced by the bone resorbing cells-osteoclasts-is promoted by osteoclast-derived EV. The EV induced axonal growth is achieved by targeting epidermal growth factor receptor (EGFR)/ErbB2 signaling/protein kinase C phosphorylation in sensory neurons. In addition, our data also indicate that osteoclasts promote sensory neurons electrophysiological activity reflecting a possible pathway in nerve sensitization in the bone microenvironment, however this effect is EV independent. CONCLUSIONS: Overall, these results identify a new mechanism of sensory bone innervation regulation and shed the light on the role of osteoclast-derived EV in shaping/guiding bone sensory innervation. These findings provide opportunities for exploitation of osteoclast-derived EV based strategies to prevent and/or mitigate pathological uncontrolled bone innervation.

Rewired glycosylation activity promotes scarless regeneration and functional recovery in spiny mice after complete spinal cord transection.

Regeneration of adult mammalian central nervous system (CNS) axons is abortive, resulting in inability to recover function after CNS lesion, including spinal cord injury (SCI). Here, we show that the spiny mouse (Acomys) is an exception to other mammals, being capable of spontaneous and fast restoration of function after severe SCI, re-establishing hind limb coordination. Remarkably, Acomys assembles a scarless pro-regenerative tissue at the injury site, providing a unique structural continuity of the initial spinal cord geometry. The Acomys SCI site shows robust axon regeneration of multiple tracts, synapse formation, and electrophysiological signal propagation. Transcriptomic analysis of the spinal cord following transcriptome reconstruction revealed that Acomys rewires glycosylation biosynthetic pathways, culminating in a specific pro-regenerative proteoglycan signature at SCI site. Our work uncovers that a glycosylation switch is critical for axon regeneration after SCI and identifies ß3gnt7, a crucial enzyme of keratan sulfate biosynthesis, as an enhancer of axon growth.

Microfluidic-based models to address the bone marrow metastatic niche complexity.

Bone marrow (BM) is a preferential metastatic site for solid cancers, contributing to higher morbidity and mortality among millions of oncologic patients worldwide. There are no current efficient therapies to minimize this health burden. Microfluidic based in vitro models emerge as powerful alternatives to animal testing, as well as promising tools for the development of personalized medicine solutions. The complexity associated with the BM metastatic niche originated a wide variety of microfluidic platforms designed to mimic this microenvironment. This review gathers the essential parameters to design an accurate in vitro microfluidic device, based on a comparative analysis of existing models created to address the different steps of the metastatic cascade.

Obesity and smoking as risk factors for invasive mechanical ventilation in COVID-19: A retrospective, observational cohort study.

PURPOSE: To describe the trajectory of respiratory failure in COVID-19 and explore factors associated with risk of invasive mechanical ventilation (IMV). MATERIALS AND METHODS: A retrospective, observational cohort study of 112 inpatient adults diagnosed with COVID-19 between March 12 and April 16, 2020. Data were manually extracted from electronic medical records. Multivariable and Univariable regression were used to evaluate association between baseline characteristics, initial serum markers and the outcome of IMV. RESULTS: Our cohort had median age of 61 (IQR 45-74) and was 66% male. In-hospital mortality was 6% (7/112). ICU mortality was 12.8% (6/47), and 18% (5/28) for those requiring IMV. Obesity (OR 5.82, CI 1.74-19.48), former (OR 8.06, CI 1.51-43.06) and current smoking status (OR 10.33, CI 1.43-74.67) were associated with IMV after adjusting for age, sex, and high prevalence comorbidities by multivariable analysis. Initial absolute lymphocyte count (OR 0.33, CI 0.11-0.96), procalcitonin (OR 1.27, CI 1.02-1.57), IL-6 (OR 1.17, CI 1.03-1.33), ferritin (OR 1.05, CI 1.005-1.11), LDH (OR 1.57, 95% CI 1.13-2.17) and CRP (OR 1.13, CI 1.06-1.21), were associated with IMV by univariate analysis. CONCLUSIONS: Obesity, smoking history, and elevated inflammatory markers were associated with increased need for IMV in patients with COVID-19.

Obesity and Smoking as Risk Factors for Invasive Mechanical Ventilation in COVID-19: a Retrospective, Observational Cohort Study.

PURPOSE: To describe the trajectory of respiratory failure in COVID-19 and explore factors associated with risk of invasive mechanical ventilation (IMV). MATERIALS AND METHODS: A retrospective, observational cohort study of 112 inpatient adults diagnosed with COVID-19 between March 12 and April 16, 2020. Data were manually extracted from electronic medical records. Multivariable and Univariable regression were used to evaluate association between baseline characteristics, initial serum markers and the outcome of IMV. RESULTS: Our cohort had median age of 61 (IQR 45-74) and was 66% male. In-hospital mortality was 6% (7/112). ICU mortality was 12.8% (6/47), and 18% (5/28) for those requiring IMV. Obesity (OR 5.82, CI 1.74-19.48), former (OR 8.06, CI 1.51-43.06) and current smoking status (OR 10.33, CI 1.43-74.67) were associated with IMV after adjusting for age, sex, and high prevalence comorbidities by multivariable analysis. Initial absolute lymphocyte count (OR 0.33, CI 0.11-0.96), procalcitonin (OR 1.27, CI 1.02-1.57), IL-6 (OR 1.17, CI 1.03-1.33), ferritin (OR 1.05, CI 1.005-1.11), LDH (OR 1.57, 95% CI 1.13-2.17) and CRP (OR 1.13, CI 1.06-1.21), were associated with IMV by univariate analysis. CONCLUSIONS: Obesity, smoking history, and elevated inflammatory markers were associated with increased need for IMV in patients with COVID-19.

The activating transcription factor 2: an influencer of cancer progression.

In contrast to the continuous increase in survival rates for many cancer entities, colorectal cancer (CRC) and pancreatic cancer are predicted to be ranked among the top 3 cancer-related deaths in the European Union by 2025. Especially, fighting metastasis still constitutes an obstacle to be overcome in CRC and pancreatic cancer. As described by Fearon and Vogelstein, the development of CRC is based on sequential mutations leading to the activation of proto-oncogenes and the inactivation of tumour suppressor genes. In pancreatic cancer, genetic alterations also attribute to tumour development and progression. Recent findings have identified new potentially important transcription factors in CRC, among those the activating transcription factor 2 (ATF2). ATF2 is a basic leucine zipper protein and is involved in physiological and developmental processes, as well as in tumorigenesis. The mutation burden of ATF2 in CRC and pancreatic cancer is rather negligible; however, previous studies in other tumours indicated that ATF2 expression level and subcellular localisation impact tumour progression and patient prognosis. In a tissue- and stimulus-dependent manner, ATF2 is activated by upstream kinases, dimerises and induces target gene expression. Dependent on its dimerisation partner, ATF2 homodimers or heterodimers bind to cAMP-response elements or activator protein 1 consensus motifs. Pioneering work has been performed in melanoma in which the dual role of ATF2 is best understood. Even though there is increasing interest in ATF2 recently, only little is known about its involvement in CRC and pancreatic cancer. In this review, we summarise the current understanding of the underestimated 'cancer gene chameleon' ATF2 in apoptosis, epithelial-to-mesenchymal transition and microRNA regulation and highlight its functions in CRC and pancreatic cancer. We further provide a novel ATF2 3D structure with key phosphorylation sites and an updated overview of all so-far available mouse models to study ATF2 in vivo.

Bio-based products control black rot (Xanthomonas campestris pv. campestris) and increase the nutraceutical and antioxidant components in kale.

Black rot of crucifers, (Xanthomonas campestris pv. campestris) is the principal yield-limiting and destructive pathogen of cruciferous crop worldwide. In order to validate a bio-based control alternative for this disease, whey, lime sulfur, biofertilizer, Bordeaux mixture or raw milk were applied to kale (Brassica oleracea var. acephala) plants. The disease control was achieved by most of the tested products. Milk-based products (raw milk and whey) and biofertilizer reduced the severity by 44 and 56% in the field. Antioxidants, crude fibber, crude protein and lipid contents and kale yield were verified in the five treatments on the leaves with and without X. campestris pv. campestris inoculation. In the absence of the pathogen (non-inoculated), lime sulfur and Bordeaux mixture improved plant nutritional value compared to organic treatments, nevertheless milk-based products and biofertilizer improved the evaluated variables more than the control. However, on leaves inoculated with X. campestris pv. campestris raw milk increased antioxidant activity, crude protein and fiber contents, whereas biofertilizer increased kale yield, lipid and antioxidant contents. Milk-based products and biofertilizer were further evaluated in greenhouse trials to determinate the activity of defense-related enzymes and lignin content. Biofertilizer treatment resulted in increased phenylalanine ammonia lyase, catalase, peroxidase activities and lignin content. Hence, the application of milk-based products and biofertilizer are promising to control black rot of crucifers and also improves food quality by boosting nutritional values and antioxidant activity.

Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface.

Junctional adhesion molecule-A (JAM-A) is a tight junction-associated signaling protein that regulates epithelial cell proliferation, migration, and barrier function. JAM-A dimerization on a common cell surface (in cis) has been shown to regulate cell migration, and evidence suggests that JAM-A may form homodimers between cells (in trans). Indeed, transfection experiments revealed accumulation of JAM-A at sites between transfected cells, which was lost in cells expressing cis- or predicted trans-dimerization null mutants. Of importance, microspheres coated with JAM-A containing alanine substitutions to residues 43NNP45 (NNP-JAM-A) within the predicted trans-dimerization site did not aggregate. In contrast, beads coated with cis-null JAM-A demonstrated enhanced clustering similar to that observed with wild-type (WT) JAM-A. In addition, atomic force microscopy revealed decreased association forces in NNP-JAM-A compared with WT and cis-null JAM-A. Assessment of effects of JAM-A dimerization on cell signaling revealed that expression of trans- but not cis-null JAM-A mutants decreased Rap2 activity. Furthermore, confluent cells, which enable trans-dimerization, had enhanced Rap2 activity. Taken together, these results suggest that trans-dimerization of JAM-A occurs at a unique site and with different affinity compared with dimerization in cis. Trans-dimerization of JAM-A may thus act as a barrier-inducing molecular switch that is activated when cells become confluent.

JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function.

Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional adhesion molecule-A (JAM-A) is a TJ-associated protein that regulates barrier; however, mechanisms linking JAM-A to epithelial permeability are poorly understood. Here we report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, small interfering RNA-mediated down-regulation of the foregoing regulatory proteins results in enhanced permeability similar to that observed after JAM-A loss. JAM-A-deficient mice and cultured epithelial cells demonstrate enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggest that JAM-A does not regulate actin turnover but modulates activity of RhoA and phosphorylation of nonmuscle myosin, both implicated in actomyosin contraction. These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.

Eating quality of "Vitela Tradicional do Montado"-PGI veal and Mertolenga-PDO veal and beef.

Physicochemical and sensory characteristics were measured in veal and beef from the Portuguese Mertolenga breed having 3 quality labels as follows: Mertolenga-PDO beef and veal which apply to purebred animals and "Vitela Tradicional do Montado"-PGI veal which applies to crossbred animals. Measurements were made in longissimus lumborum muscle aged for 6days. The temperature 3h post-mortem (T3), cooking losses and Warner-Bratzler shear force (WBSF) reflected carcass weight (CW) differences between groups. The pigment content was influenced by age, with beef having higher values than veal. WBSF correlated negatively with intramuscular fat in Mertolenga-PDO beef, but not on veal. WBSF correlated positively with cooking losses and negatively with myofibrillar fragmentation index, tenderness, juiciness and overall acceptability. Cooking losses and juiciness were the main contributors for the tenderness differences. Vitela Tradicional do Montado-PGI and Mertolenga-PDO veal had lighter colour and were considered tender. The three meat types were well discriminated based on pHu, a* and C* parameters by canonical discriminant analysis.

Intracellular mediators of JAM-A-dependent epithelial barrier function.

Junctional adhesion molecule-A (JAM-A) is a critical signaling component of the apical junctional complex, a structure composed of several transmembrane and scaffold molecules that controls the passage of nutrients and solutes across epithelial surfaces. Observations from JAM-A-deficient epithelial cells and JAM-A knockout animals indicate that JAM-A is an important regulator of epithelial paracellular permeability; however, the mechanism(s) linking JAM-A to barrier function are not understood. This review highlights recent findings relevant to JAM-A-mediated regulation of epithelial permeability, focusing on the role of upstream and downstream signaling candidates. We draw on what is known about proteins reported to associate with JAM-A in other pathways and on known modulators of barrier function to propose candidate effectors that may mediate JAM-A regulation of epithelial paracellular permeability. Further investigation of pathways highlighted in this review may provide ideas for novel therapeutics that target debilitating conditions associated with barrier dysfunction, such as inflammatory bowel disease.

A brief report on some health aspects of rats fed with crescent levels of recombinant chagasin, a potential plant defense protein.

Chagasin may be considered a potential plant-incorporated protectant (PIP) protein due to its deleterious effects on insect pests. However, extensive safety studies with PIP's are necessary before introducing them into the target plant. Thus, a short-term feeding trial in rats with high doses of r-chagasin was conducted to provide evidences about its safety. Three test diets containing casein + r-chagasin (0.25, 0.5 and 1% of total protein) were offered to rats (10 days). The test diets did not show adverse effects upon the development, organ weight, hematological parameters and serum protein profiles of rats, providing preliminary information on the safety of r-chagasin.

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour.

AIMS: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness. The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. METHODS AND RESULTS: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. CONCLUSIONS: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Phencyclidine and dizocilpine induced behaviors reduced by N-acetylaspartylglutamate peptidase inhibition via metabotropic glutamate receptors.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptor open channel blockers phencyclidine (PCP) and dizocilpine (MK-801) elicit schizophrenia-like symptoms in humans and in animal models. Group II metabotropic glutamate receptor agonists reverse the behavioral effects of PCP and MK-801 in animal models. N-acetylaspartylglutamate (NAAG), the third most prevalent neurotransmitter in the mammalian nervous system, is a selective group II metabotropic glutamate receptor agonist. We previously reported that ZJ43, a potent inhibitor of the enzymes that inactivate synaptically released NAAG, reduced motor and stereotypic effects of PCP in the rat. METHODS: To confirm the efficacy of NAAG peptidase inhibition in decreasing motor behaviors induced by PCP and MK-801, ZJ43 was tested in additional schizophrenia models. RESULTS: ZJ43 reduced MK-801-induced motor activation in a mouse model that has been used to characterize the efficacy of a wide range of pharmacotherapies for this human disorder. In a second mouse strain, the peptidase inhibitor reduced PCP-induced stereotypic movements. ZJ43 also reduced PCP-induced negative symptoms in a resident-intruder assay. The group II metabotropic glutamate receptor antagonist, LY341495, blocked the effect of NAAG peptidase inhibition in these mouse models of positive and negative PCP- and MK-801-induced behaviors. Additionally, LY341495 alone increased some PCP-induced behaviors suggesting that normal levels of NAAG act to moderate the effect of PCP via a group II mGluR. CONCLUSIONS: These data support the proposal that NAAG peptidase inhibition and elevation of synaptic NAAG levels represent a new therapeutic approach to treating the positive and negative symptoms of schizophrenia that are modeled by open channel NMDA receptor antagonists.

Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests.

Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.