Integrating system biology and intratumor gene therapy by trans-complementing the appropriate co-stimulatory molecule as payload in oncolytic herpes virus.

Systems biology has been applied at the multi-scale level within the cancer field, improving cancer prevention, diagnosis and enabling precision medicine approaches. While systems biology can expand the knowledge and skills for oncological treatment, it also represents a challenging expedition due to cancer complexity, heterogeneity and diversity not only between different cancer indications, but also in its evolution process through space and time. Here, by characterizing the transcriptional perturbations of the tumor microenvironment induced by oncolytic, we aimed to rationally design a novel armed oncolytic herpes virus. We found that intratumor oncovirotherapy with HSV-1 induces T-cell activation signatures and transcriptionally activates several costimulatory molecules. We identified differentially expressed costimulatory receptors and binding partners, where inducible co-stimulators (ICOS) resulted in the potentially most beneficial targeted therapy. Through an ex-vivo transcriptomic analysis, we explored the potential of arming an oncolytic virus as a combination therapy strategy; in particular, we engineered a targeted herpes virus encoding ICOSL (THV_ICOSL), which resulted in a significant improvement in tumor size control compared to unarmed parental virus. Also, combination with a PD-1 inhibitor enhanced antitumor efficacy as predictable by upregulation of PD-1 and ligands pair (PD-L1/PD-L2) upon oncolytic virus injection. Generation of the human version of this virus encoding hICOSL orthologue effectively and specifically activated human T cells by triggering the ICOS pathway. Our data support the data-driven generation of armed oncolytic viruses as combination immunotherapeutic with checkpoint inhibitors.

In vivo immobilized carbonic anhydrase and its effect on the enhancement of CO2 absorption rate.

Reactive absorption into aqueous solutions promoted by carbonic anhydrase (CA, E.C. 4.2.1.1.) has been often proposed as a post-combustion CO2 capture process. The state of the art reveals the need for efficient biocatalyst based on carbonic anhydrase that can be used to further develop CO2 capture and utilization technologies. The present study is focused on the use of a thermostable CA-based biocatalyst. The carbonic anhydrase SspCA, from the thermophilic bacterium Sulfurihydrogenibium yellowstonense, was in vivo immobilized as membrane-anchored protein (INPN-SspCA) on the outer membrane of Escherichia coli cells. The dispersed biocatalyst, made by cell membrane debris, was characterized in terms of its contribution to the enhancement of CO2 absorption in carbonate/bicarbonate alkaline buffer at operating conditions relevant for industrial CO2 capture processes. The amount of immobilized enzyme, estimated by SDS-PAGE, resulted in about 1 mg enzyme/g membrane debris. The apparent kinetics of the biocatalyst was characterized through CO2 absorption tests in a stirred cell lab-scale reactor assuming a pseudo-homogeneous behaviour of the biocatalyst. At 298 K, the assessed values of the second-order kinetic constant ranged between 0.176 and 0.555 L∙mg-1∙s-1. Reusability of the biocatalyst after 24 h showed the absence of free enzyme release in the alkaline solvent. Moreover, the equilibration of dispersed cell membrane debris against the alkaline buffer positively affected the performances of the heterogeneous biocatalyst. These results encourage further studies on the in vivo immobilized SspCA aimed at optimizing the enzyme loading on the cell membrane and the handling of the biocatalyst in the CO2 absorption reactors.

Oncolytic vaccines increase the response to PD-L1 blockade in immunogenic and poorly immunogenic tumors.

Activation of immune checkpoint pathways and limited T- cell infiltration result in immunological escape of tumors. Although immune checkpoint inhibitors are currently approved for several types of cancers, the response rate is often limited by the lack of tumor specific T-cells within the malignant tissue. Therefore, new combinatorial strategies are needed to enhance the clinical benefit of immune checkpoint inhibitors. We have previously developed PeptiCRAd, an oncolytic vaccine platform capable of directing the immune response toward tumor epitopes. In this study, we evaluated whether the platform could be used to increase the response rate to checkpoint inhibitors in both highly immunogenic and poorly immunogenic tumors, such as melanoma and triple negative breast cancer (TNBC). We report here that anti-PD-L1 therapy in combination with PeptiCRAd significantly reduced the growth of melanomas and increased the response rate to checkpoint inhibition. In fact, we registered a higher rate of complete responses among mice treated with the combination. This approach promoted the presence of non-exhausted antigen-specific T-cells within the tumor in comparison to anti-PD-L1 monotherapy. Furthermore, we found that targeting both MHC-I and II restricted tumor epitopes was necessary to decrease the growth of the poorly immunogenic TNBC model 4T1 and that combination with PD-L1 blockade increased the number of responders to checkpoint inhibition. Finally, the described strategy was validated in a translational in vitro model using HLA matched human PBMCs and tumor cell lines. Consistent to our previous results, improved cytotoxicity was observed with combination of PeptiCRAd and anti-PD-L1. These results demonstrate that oncolytic virus based cancer vaccine can significantly improve the response rate to checkpoint blocking antibodies in the context of immunogenic and non-immunogenic tumors.

Antitumor effect of oncolytic virus and paclitaxel encapsulated in extracellular vesicles for lung cancer treatment.

Standard of care for cancer is commonly a combination of surgery with radiotherapy or chemoradiotherapy. However, in some advanced cancer patients this approach might still remaininefficient and may cause many side effects, including severe complications and even death. Oncolytic viruses exhibit different anti-cancer mechanisms compared with conventional therapies, allowing the possibility for improved effect in cancer therapy. Chemotherapeutics combined with oncolytic viruses exhibit stronger cytotoxic responses and oncolysis. Here, we have investigated the systemic delivery of the oncolytic adenovirus and paclitaxel encapsulated in extracellular vesicles (EV) formulation that, in vitro, significantly increased the transduction ratio and the infectious titer when compared with the virus and paclitaxel alone. We demonstrated that the obtained EV formulation reduced the in vivo tumor growth in animal xenograft model of human lung cancer. Indeed, we found that combined treatment of oncolytic adenovirus and paclitaxel encapsulated in EV has enhanced anticancer effects both in vitro and in vivo in lung cancer models. Transcriptomic comparison carried out on the explanted xenografts from the different treatment groups revealed that only 5.3% of the differentially expressed genes were overlapping indicating that a de novo genetic program is triggered by the presence of the encapsulated paclitaxel: this novel genetic program might be responsible of the observed enhanced antitumor effect. Our work provides a promising approach combining anticancer drugs and viral therapies by intravenous EV delivery as a strategy for the lung cancer treatment.

An Overview of the Selectivity and Efficiency of the Bacterial Carbonic Anhydrase Inhibitors.

The possibility to develop new antibacterial agents raised much interest recently. The main classes of antibiotics clinically used nowadays act towards the inhibition of four classical targets: a) cell wall biosynthesis; b) protein biosynthesis; c) DNA and RNA biosynthesis; d) folate biosynthesis. Recently, carbonic anhydrases (CAs, EC 4.2.1.1) started to be investigated in detail in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many bacteria, CAs are essential for the life cycle of the organism and that their inhibition leads to growth impairment or growth defects of the pathogen. CAs catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. Several classes of CA inhibitors (CAIs) are known to date: the metal complexing anions and the unsubstituted sulfonamides, which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere, generating trigonal- bipyramidal species are the classical, most frequently investigated ones. In many cases effective inhibitors were detected, some of which also inhibited the bacterial growth in vivo. However, very few of the detected inhibitors were also selective for the bacterial over the human, off target isoforms such as hCA II. Using structure-based drug design processes, we estimate that it will be possible to achieve the desired selectivity for inhibiting preferentially the bacterial but not the host CA isoforms.

Toxicity, accumulation, and removal of heavy metals by three aquatic macrophytes.

A comprehensive understanding of the uptake, tolerance, and transport of heavy metals by plants will be essential for the development of phytoremediation technologies. In the present paper, we investigated accumulation, tissue and intracellular localization, and toxic effects of cadmium (Cd), lead (Pb), zinc (Zn), and copper (Cu) in three aquatic macrophytes (the angiosperms Lemna minor and Elodea canadensis, and the moss Leptodictyum riparium). We also tested and compared their capacity to absorb heavy metal from water under laboratory conditions. Our data showed that all the three species examined could be considered good bioaccumulators for the heavy metals tested. L. riparium was the most resistant species and the most effective in accumulating Cu, Zn, and Pb, whereas L. minor was the most effective in accumulating Cd. Cd was the most toxic metal, followed by Pb, Cu, and Zn. At the ultrastructural level, sublethal concentrations of the heavy metals tested caused induced cell plasmolysis and alterations of the chloroplast arrangement. Heavy metal removal experiments revealed that the three macrophytes showed excellent performance in removing the selected metals from the solutions in which they are maintained, thus suggesting that they could be considered good candidates for wastewaters remediation purpose.

Gene expression profiling of phytoplasma-infected Madagascar periwinkle leaves using differential display.

Phytoplasmas are small (0.2-0.8 µm), wall-less, pleiomorphic prokaryotes responsible of numerous economically important plant diseases. They are characterized by a very small genome and are obligate parasites of phloem tissues and some insects that act as vectors of infection. To investigate molecular mechanisms involved in pathogenesis, the differential display technique was here applied to identify plant genes whose transcription was significantly altered in leaves of Madagascar periwinkle (Catharanthus roseus (L.) G.Don) infected by 'Candidatus Phytoplasma pyri'. We detected, reamplified, cloned, and sequenced 16 putative differentially expressed cDNA fragments. Northern blot analysis revealed that seven of the 16 genes identified were up-regulated following phytoplasma infection, while three genes were down-regulated. The remaining six genes did not show significant changes in the level of expression. Identified genes are mainly involved in plant defence/stress responses, protein metabolism and transport, transcriptional regulation, vesicle trafficking, and carbohydrate metabolism. The possible role played by these genes in the phytoplasma infection is discussed.

Dietary sodium intake in a sample of adult male population in southern Italy: results of the Olivetti Heart Study.

BACKGROUND/OBJECTIVES: To assess dietary habitual sodium intake and the association between daily sodium intake and anthropometric indices, food habits and hypertension in the sample of adult male population participating in the Olivetti Heart Study. SUBJECTS/METHODS: The study population was composed of 940 men participating in the 2002-2004 follow-up examination of the Olivetti Heart Study. Blood pressure, anthropometric indices, biochemical parameters and sodium excretion in a 24-h urine collection were measured. The frequency of consumption of selected foods was estimated by a food frequency questionnaire (FFQ) capturing the previous year data. In a subgroup of the study population (n=138), the fractional excretion of sodium was estimated by endogenous lithium clearance. RESULTS: Dietary sodium intake estimated by 24 h urinary excretion was 203+/-70 mmol/day. Sodium excretion was significantly lower in treated hypertensive patients and higher in overweight/obese participants when compared with normotensive and normal-weight individuals, respectively. In addition, the inverse correlation detected in normal-weight individuals (r=-0.321; P<0.05) between fractional proximal tubular sodium reabsorption and dietary sodium intake was disrupted in overweight/obese individuals (r=0.058; P=NS). The independent determinants of 24 h urinary sodium excretion were body mass index (BMI), the occurrence of antihypertensive treatment, and frequency of consumption of pasta and cold cuts. CONCLUSIONS: Habitual salt intake in this sample of male adult population in southern Italy was well above the recommended amounts. A higher salt intake and an altered renal sodium handling were observed in overweight and obese participants. Sodium intake was only slightly reduced in hypertensive participants on pharmacological therapy.

Accumulation, localisation, and toxic effects of cadmium in the liverwort Lunularia cruciata.

Accumulation, tissue and intracellular localisation, and toxic effects of cadmium were investigated in the liverwort Lunularia cruciata. The results of analyses carried out by atomic absorption spectrometry on single plants showed that the cadmium accumulation was dose- and time-dependent. Cadmium localisation was assessed by X-ray scanning electron microscopy microanalysis in gemmalings and in the different tissues of the thallus and by X-ray transmission electron microscopy microanalysis at the cellular level. The metal preferentially accumulated in the hyaline parenchyma and at the base of the gemma cups. Inside the cell, cadmium accumulated in the vacuoles and the cell wall. Metal accumulation was accompanied by a concomitant increase in sulphur content within the vacuoles of stressed cells. Gel-permeation chromatography showed that most of the cadmium was associated with a low-molecular-mass fraction eluting at a ratio of elution volume to void volume corresponding to that of phytochelatins. The excess of sulphur deposited in the vacuoles may well have been caused by the stress-induced synthesis of phytochelatins. At the ultrastructural level, sublethal concentrations of cadmium caused alterations of the fine structure of the cells, inducing marked alterations of the chloroplast structure. Cadmium also induced a dose-dependent inhibition of apical thallus growth and gemma germination.

Identification of cadmium-sensitive genes in the Antarctic fish Chionodraco hamatus by messenger RNA differential display.

To investigate the ability of cadmium to affect gene transcription in fish, the messenger RNA (mRNA) differential display technique was used to analyze gene expression in the Antarctic icefish Chionodraco hamatus exposed to sublethal doses of cadmium salt. Seven DNA complementary to RNA (cDNA) bands whose steady-state levels of expression significantly changed in response to cadmium exposure were identified. The results obtained show that two groups of genes are affected by cadmium in icefish liver. The first group comprises genes that are up-regulated by the metal: in particular, a gene encoding the heat-shock protein HSP70 and another encoding a protein homologous to GP49 of Sparus aurata egg envelope. The other group comprises genes down-regulated by cadmium. These are the transferrin gene and a gene encoding a protein presenting homology to mouse T2K, a kinase having a role in the prevention of apoptosis. Three cDNAs had no homology to known gene sequences, thus suggesting that may either encode not yet identified proteins, or correspond to untranslated regions of mRNA molecules.

Structural and functional analysis of metal regulatory elements in the promoter region of genes encoding metallothionein isoforms in the Antarctic fish Chionodraco hamatus (icefish).

To investigate the regulation of Chionodraco hamatus metallothionein (MT) encoding genes about 1000-bp regions of both MT-I and MT-II gene promoters were cloned and sequenced. Both promoters were rich in A-T content, and lacked the canonical TATA box; several putative cis-regulatory sequences were also present. In the MT-I promoter, four MREs were identified within the first 300 bp from the ATG codon. In the MT-II promoter, seven MREs were organized into two clusters, one containing three MREs located close to the ATG codon, and the other consisting of four MREs lying 500-900 bp upstream of the transcription starting point. The alignment of the MT-I and MT-II promoter regions showed 57% identity, which increased to 87% in the 300-bp region upstream of the ATG. Only the three proximal putative MREs identified were conserved both in position and sequence. Functional analysis of MT-I and MT-II promoters was performed by introducing deletion mutants of the 5'-flanking regions into vector pGL-3, directly upstream of the firefly luciferase reporter gene. Each construct was tested in the HepG2 cell lines in the absence or presence of zinc or cadmium ions. Maximum inducibility of the MT-II gene promoter was achieved with a construct containing both the proximal and the distal MRE clusters. The lack of the most distally located MRE dramatically affected MT-II promoter sensitivity to metals; removal of the distal cluster of MREs also reduced metal inducibility. The MT-I promoter was more compact, since maximal activity and metal inducibility depended on the presence of the proximal cluster of four MREs. This study suggests that the different organization of the MT-I and MT-II gene promoter regions might account for the observed differences in the basal and metal-induced expression of MT-I and MT-II isoforms in the C. hamatus liver.

Structural characterization and thermal stability of Notothenia coriiceps metallothionein.

Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps, and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs. However, NMR spectroscopy shows that the dynamic behaviour of the two domains is markedly different. With the aid of absorption and CD spectroscopies, we studied the conformational and electronic features of fish and mouse recombinant Cd-MT and the changes produced in these proteins by heating. When the temperature was increased from 20 to 90 degrees C, the Cd-thiolate chromophore absorbance at 254 nm of mouse MT was not modified up to 60 degrees C, whereas the absorbance of fish MT decreased significantly starting from 30 degrees C. The CD spectra also changed quite considerably with temperature, with a gradual decrease of the positive band at 260 nm that was more pronounced for fish than for mouse MT. The differential effect of temperature on fish and mouse MTs may reflect a different stability of metal-thiolate clusters of the two proteins. Such a conclusion is also corroborated by results showing differences in metal mobility between fish and mouse Zn-MT.

Susceptibility to heavy metals and cadmium accumulation in aerobic and anaerobic thermophilic microorganisms isolated from deep-sea hydrothermal vents.

Thirty thermophilic strains isolated from heavy metal-rich hydrothermal vent sites at Lau Basin were tested for their susceptibility to cadmium, zinc, cobalt, and nickel. The 14 aerobic spore formers belonging to the genus Bacillus, 6 anaerobic fermenters from the order Thermotogales, and 10 anaerobic sulfur reducers from the order Thermococcales could be clearly distinguished according to their metal susceptibilities. The Thermococcales were found to exhibit the highest resistance to cadmium and zinc, whereas Thermotogales were highly sensitive to these metals. In contrast, the Thermotogales displayed the highest resistance to cobalt ions. No clear distinction could be established between the metal susceptibilities of these strains and seven reference organisms used for comparative studies. Cadmium resistance, slightly inducible in some cadmium-resistant bacilli, was not plasmid mediated. The amount of cadmium immobilized by the Thermotogales was related to their level of resistance to this metal.

Metallothioneins in antarctic fish: evidence for independent duplication and gene conversion.

In the present paper, we examine eight species of Antarctic fish belonging to the suborder Notothenioidei, using reverse-transcriptase polymerase chain reaction, to investigate the presence of mRNAs encoding metallothionein (MT) isoforms. A total of 168 bp from the coding region and the complete (133-165 bp) 3' untranslated region (UTR) was obtained for all species (for three of them, we also sequenced the full-length cDNA, including the 5' UTR). Phylogenetic analyses carried out on the MT-coding region suggest monophyly for Antarctic fish MTs with respect to other teleost MT genes. Analyses also revealed that notothenioid MTs can be divided into at least two groups of paralogy, MT-1 and MT-2. These results indicate that notothenioid MT isoforms arose from at least one gene duplication event occurring in the ancestral lineage of the Notothenioidei. This duplication occurred independent of the one which gave origin to two metallothionein isoforms in the rainbow trout. In addition, an instance of gene conversion was observed between MT-1 and MT-2 genes in Notothenia coriiceps. Analyses of the 5' UTR, combined with quantitative assay of differential expression of MT-1 and MT-2, indicate that only the 3' UTR underwent a gene conversion event in the mentioned species. These findings, together with the observation of a differential pattern of expression for the two MT isoforms, disclose an unexpected complexity in the evolution and function of notothenioid MTs; as in most teleost species examined (apart from the rainbow trout), a single MT form is present.

Cathepsin D from the liver of the antarctic icefish Chionodraco hamatus exhibits unusual activity and stability at high temperatures1.

Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion-exchange chromatography followed by affinity chromatography on concanavalin-A Sepharose. The purified enzyme showed a molecular mass of 40 kDa and displayed optimal activity at pH 3.0 with a synthetic chromogenic substrate. The N-terminal sequence of this proteinase was determined by automated Edman degradation and was used to design a primer for use in reverse-transcriptase polymerase chain reaction. The open reading frame of the cloned cDNA encoded an aspartic proteinase, which contained the experimentally determined N-terminal sequence. The predicted sequence (396 residues) had a high similarity with those of cathepsin D from various vertebrate sources, but was considerably different from that of nothepsin, a distinct aspartic proteinase described previously from Antarctic fish [1]. Determination of kinetic parameters for substrate hydrolysis showed that, at temperatures between 8 and 50 degrees C, the icefish cathepsin D had a higher specificity constant (kcat/Km) than human cathepsin D. The stability of both enzymes was measured at 50 degrees C and half-lives of 55 and 3 min were derived for icefish and human cathepsin D, respectively.

Molecular cloning and sequence determination of a novel aspartic proteinase from Antarctic fish.

In the present report, we describe a novel aspartic proteinase from the liver of two Antarctic fish species. The nucleotide sequences of the cDNA obtained from the two fishes show 90% identity with each other but only 58% identity with aspartic proteinases from other sources. Sequence analysis shows features for the Antarctic enzymes which are not present in related enzymes of other organisms.

Cadmium-induced differential accumulation of metallothionein isoforms in the Antarctic icefish, which exhibits no basal metallothionein protein but high endogenous mRNA levels.

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.

PCR amplification and cloning of metallothionein complementary DNAs in temperate and Antarctic sea urchin characterized by a large difference in egg metallothionein content.

Metallothionein levels were determined in the eggs of two sea urchin species, the Mediterranean Sphaerechinus granularis and the Antarctic Sterechinus neumayeri. While appreciable levels of metallothionein were found in S. granularis eggs, a negligible amount was detected in S. neumayeri. Two metallothionein isoforms were purified from S. granularis, and metallothionein cDNAs were obtained by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Two distinct cDNA species were cloned and sequenced. The translated amino acid sequences of these two forms consisted of 67 residues and differed in two amino acid substitutions. Despite the lack of metallothionein in S. neumayeri eggs, a metallothionein cDNA was obtained by RT-PCR amplification and a single amino acid sequence coding for a 63 residues MT was deduced. A comparative analysis of the primary structure of S. granularis and S. neumayeri metallothioneins with those of the other sea urchin metallothioneins has been performed. Sea urchin metallothioneins appear to be less similar to each other than metallothioneins of closely related vertebrates.

Difference in hepatic metallothionein content in Antarctic red-blooded and haemoglobinless fish: undetectable metallothionein levels in haemoglobinless fish is accompanied by accumulation of untranslated metallothionein mRNA.

Icefish (family Channichthyidae, suborder Nothothenioidei) are a group of Antarctic fish that have evolved unique phenotypes in order to adapt to the environment in which they live. Besides the lack of haemoglobin and the drastic reduction in the number of erythrocyte-like cells, another striking feature of the icefish is that their liver is devoid of metallothionein. These cysteine-rich heavy-metal-binding proteins are usually present in large amounts in a large variety of organisms, from bacteria to mammals. Despite the failure to detect appreciable levels of metallothionein in icefish liver, a cDNA encoding metallothionein was produced from total RNA by reverse transcriptase PCR. The icefish metallothionein showed high percentage identity with metallothionein from Trematomus bernachii, a red-blooded Antarctic fish in which a normal content of hepatic metallothionein was found. Steady-state mRNA levels were assessed in fish liver by high-stringency hybridization of the metallothionein probe with total RNA. The results showed that icefish livers retain large amounts of untranslated metallothionein mRNA. The stability of the icefish transcript might be correlated with the lack of specific motifs in the untranslated 3' ends of mRNA.

Crystal structure of the bovine alpha-chymotrypsin:Kunitz inhibitor complex. An example of multiple protein:protein recognition sites.

The crystal structure of bovine alpha-chymotrypsin (alpha-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 A resolution (R-factor = 0.18). The proteinase:inhibitor complex forms a compact dimer (two alpha-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between alpha-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P1 residue of BPTI, however, does not occupy the alpha-CHT S1 specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217.