Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40

Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-α-L-galactopyranosyl-(1→3)-D-galactose] using various β-agarases. Neoagarobiose hydrolase is an enzyme that acts on the α-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 Å, β = 101.86°. The crystals diffracted to 1.98 Å resolution and possibly contains two molecules in the asymmetric unit

Bonghan Ducts as Possible Pathways for Cancer Metastasis

Objective: The present study has been designed to find a possible new route for the metastasis of cancer cells on the fascia surrounding tumor tissue using a novel technique of trypan blue staining. Materials and Methods: Tumor tissues were grown in the skin of nude mice after subcutaneous inoculation with human lung cancer cells. Trypan blue was recently identified as a dye with specificity for Bonghan ducts (BHDs) and not other tissues, such as blood or lymph vessels or nerves. Results: We demonstrate that the trypan blue staining technique allows the first visualization of BHDs which are connected to tumor tissues

Bonghan Ducts as Possible Pathways for Cancer Metastasis

Objective: The present study has been designed to find a possible new route for the metastasis of cancer cells on the fascia surrounding tumor tissue using a novel technique of trypan blue staining. Materials and Methods: Tumor tissues were grown in the skin of nude mice after subcutaneous inoculation with human lung cancer cells. Trypan blue was recently identified as a dye with specificity for Bonghan ducts (BHDs) and not other tissues, such as blood or lymph vessels or nerves. Results: We demonstrate that the trypan blue staining technique allows the first visualization of BHDs which are connected to tumor tissues

Model-Based Complete Enzymatic Production of 3,6-Anhydro‑l‑galactose from Red Algal Biomass

3,6-Anhydro-l-galactose (l-AHG) is a bioactive constituent of agar polysaccharides. To be used as a cosmetic or pharmaceutical ingredient, l-AHG is more favorably prepared by enzymatic saccharification of agar using a combination of agarolytic enzymes. Determining the optimum enzyme combination from the natural repertoire is a bottleneck for designing an efficient enzymatic-hydrolysis process. We consider all theoretical enzymatic-saccharification routes in the natural agarolytic pathway of a marine bacterium, <i>Saccharophagus degradans</i> 2-40. Among these routes, three representative routes were determined by removing redundant enzymatic reactions. We simulated each l-AHG production route with simple kinetic models and validated the reaction feasibility with an experimental procedure. The optimal enzyme mixture (with 67.3% maximum saccharification yield) was composed of endotype β-agarase, exotype β-agarase, agarooligosaccharolytic β-galactosidase, and α-neoagarobiose hydrolase. This approach will reduce the time and effort needed for developing a coherent enzymatic process to produce l-AHG on a mass scale

Primary pyomyositis and uveitis in a cat

Abstract A 6‐year‐old neutered male Siamese cat was referred for investigation of hindlimb ataxia and blindness of 2 weeks’ duration. A swollen right hind limb, with no history of trauma, and no evidence of an external wound, was observed on physical examination. Ophthalmic examination revealed bilateral absence of the menace response and changes consistent with uveitis. Blood tests identified changes consistent with inflammation including serum amyloid A elevation. Infectious disease testing was negative. Degenerate neutrophils and bacterial cocci were detected on fine needle aspiration cytology of the affected limb. Thoracic radiography and abdominal ultrasonography identified no abnormalities. Primary pyomyositis was suspected and clindamycin was prescribed following Penrose drain tube placement. In addition, eye drops containing tobramycin, atropine, and prednisolone were administered. The clinical signs and serum amyloid A level were markedly improved after 5 days of treatment. Based on the medical history and lack of other findings, the uveitis was suspected to be secondary to the pyomyositis. The clinical signs resolved completely, and no recurrence was reported within a 6‐month follow‐up period. To the best of our knowledge, primary pyomyositis with uveitis has not been previously reported in cats

Preclinical Efficacy of Clumping Factor A in Prevention of Staphylococcus aureus Infection

ABSTRACT Treatment of Staphylococcus aureus infections has become increasingly difficult because of the emergence of multidrug-resistant isolates. Development of a vaccine to prevent staphylococcal infections remains a priority. To determine whether clumping factor A (ClfA) is a good target protein for inclusion in a multivalent vaccine, we evaluated its efficacy in a variety of relevant staphylococcal infection models, challenging with different S. aureus strains. ClfA adsorbed to Alhydrogel and mixed with Sigma Adjuvant System was more immunogenic and stimulated a more robust Th17 response than ClfA administered with alum alone. ClfA immunization induced the production of functional antibodies in rabbits and mice that blocked S. aureus binding to fibrinogen and were opsonic for S. aureus strains that produced little or no capsular polysaccharide. Mice immunized with ClfA showed a modest reduction in the bacterial burden recovered from subcutaneous abscesses provoked by S. aureus USA300 strain LAC. In addition, the ClfA vaccine reduced lethality in a sepsis model following challenge with strain Newman, but not ST80. Vaccination with ClfA did not protect against surgical wound infection, renal abscess formation, or bacteremia. Passive immunization with antibodies to ClfA did not protect against staphylococcal bacteremia in mice or catheter-induced endocarditis in rats. Some enhancement of bacteremia was observed by ClfA immunization or passive administration of ClfA antibodies when mice were challenged by the intraperitoneal route. Although rodent models of staphylococcal infection have their limitations, our data do not support the inclusion of ClfA in an S. aureus multivalent vaccine

Suspected Cerebral Salt Wasting Syndrome with Cervical Spinal Lesion in a Domestic Shorthair Cat

A 12-year-old spayed female domestic short cat was presented with tetraplegia. The cat also showed signs of hyponatremia and dehydration, which were rapidly corrected by intravenous fluid infusion. Based on thorough physical and neurological examinations, the patient was suspected of having an intracranial disease. MRI revealed a high-signal T2 image of the bilateral parietal cerebral cortical gray matter junction, which is associated with fast electrolyte calibration, and a high-signal T2 image of the C2 spinal cord ventral area, which is associated with ischemic myelopathy. The cat reappeared three days later due to anorexia. Laboratory examinations revealed that the cat was clinically dehydrated and exhibited hyponatremia. Other causes of hyponatremia were excluded through history-taking, laboratory examination, imaging, and therapeutic response to fluid therapy, except for cerebral salt-wasting syndrome (CSWS). The cat was discharged 3 days after the start of fludrocortisone therapy with electrolytes within the normal range. Magnetic resonance imaging (MRI) was performed again 1 month after hospitalization, and the cerebral lesion disappeared, but the spinal cord lesion worsened compared to the previous image. The patient was euthanized due to the progression of the spinal lesion, with a poor prognosis and poor quality of life. This is the first case of suspected CSWS with a cervical spinal lesion in a cat

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2–40

In agarolytic microorganisms, a-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-L-galactopyranosyl-(1,3)-D-galactose) into fermentable monosaccharides (D-galactose and 3,6-anhydro-L-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2–40 in a native protein and D-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed β-propeller catalytic domain. The structure of the enzyme–ligand (D-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways

Novel Monomeric Fungal Subtilisin Inhibitor from a Plant-Pathogenic Fungus, Choanephora cucurbitarum: Isolation and Molecular Characterization.

The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated "choanepins." Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd ]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors.IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications

Identification and characterization of a novel cancer/testis antigen gene CAGE-1

Serological analysis of cDNA expression library (SEREX) was employed to identify cancer-associated genes. By screening DNA expression libraries with sera of patients with lung cancers, we identified a total of 49 genes that specifically reacted with the sera of patients with lung cancers. Among these, we characterized a novel gene with expression pattern similar to that of cancer/testis antigens. Its open reading frame is 1920 bp in size and encodes for putative protein composed of 639 amino acids. Southern blot analysis reveals that this gene exists as single copy. In vitro transcription/translation and Western blot analysis confirm that this gene encodes a protein of 73 kDa in size. The comparison of cDNA and genomic sequences reveals that it is composed of 11 exons and 10 introns. This gene displays testis-specific expression among normal tissues, and wide expression among various cancer tissues and cancer cell lines. A study using GFP fusion construct reveals mainly nuclear localization of CAGE-1 protein. The expression of this clone is relatively higher in cancer tissues compared with their surrounding non-cancerous tissues. This suggests that overexpression of CAGE-1 may be associated with the progression of tumor. Because of its association with cancer, this gene was named cancer-associated gene-1 (CAGE-1). Given the fact that several cancer/testis antigens reportedly induce cytolytic T lymphocyte (CTL) reactions, it is reasonable that this gene. will be a valuable target for cancer immunotherapy. The exact functional role of CAGE-I in tumorigenesis remains to be seen. (C) 2002 Elsevier Science B.V All rights reserved