Ultrasensitive and rapid diagnostic tool for detection of Acanthamoeba castellanii.

Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and diagnostic delays can lead to irreversible damage to the vision. The gold standard for diagnosis has been a sample culture that lasts approximately 2 weeks. Nevertheless, the essence of time has led to the need for an accurate and fast technique to detect A. castellanii from a sample. We developed both traditional and quantitative real-time-PCR-based methods to detect A. castellanii in less than 3 hours and with the sensitivity of one amoeba. Diagnostic laboratories can select the best-suited method for their purposes from 2 comparable methods. The correct treatment can be initiated from the emergency room when the diagnosis has been made quickly within a few hours, hence saving the patient from long-term complications.

Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva.

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.

Carbonic Anhydrase VI in Skin Wound Healing Study on Car6 Knockout Mice.

Carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. CA VI is secreted to milk and saliva, and it could contribute to wound closure, as a potential trophic factor, in animals that typically lick their wounds. Our aim was to investigate whether human CA VI improves skin-wound healing in full-thickness skin-wound models. The effect was studied in Car6 -/- knockout mice and wild type littermates. Half of both mice strains were given topically administered, milk-derived CA VI after wounding and eight hours later. The amount of topically given CA VI exceeded the predicted amount of natural saliva-delivered CA VI. The healing was followed for seven days and studied from photographs and histological sections. Our results showed no significant differences between the treatment groups in wound closure, re-epithelization, or granulation tissue formation, nor did the Car6 genotype affect the healing. Our results demonstrate that CA VI does not play a major role in skin-wound healing and also suggest that saliva-derived CA VI is not responsible for the licking-associated improved wound healing in animals.

Identification and characterization of a novel zebrafish (Danio rerio) pentraxin-carbonic anhydrase.

BACKGROUND: Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15-21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. METHODS: We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI-PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI-PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. RESULTS: The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. DISCUSSION: These findings provide novel insights into the evolution, structure, and function of this unique CA form.

Altered gene expression in the lower respiratory tract of Car6 (-/-) mice.

From birth, the respiratory tract mucosa is exposed to various chemical, physical, and microbiological stress factors. Efficient defense mechanisms and strictly regulated renewal systems in the mucosa are thus required. Carbonic anhydrase VI (CA VI) is the only secreted isoenzyme of the α-CA gene family. It is transported in high concentrations in saliva and milk into the alimentary tract where it contributes to optimal pH homeostasis. Earlier study of transcriptomic responses of Car6 (-/-) mice has shown changes in the response to oxidative stress and brown fat cell differentiation in the submandibular gland. It has been suggested that CA VI delivered to the mucosal surface of the bronchiolar epithelium is an essential factor in defense and renewal of the lining epithelium. In this study, the transcriptional effects of CA VI deficiency were investigated in both trachea and lung of Car6 (-/-) mice using a cDNA microarray analysis. Functional clustering of the results indicated significant changes of gene transcription in the lower airways. The altered biological processes included antigen transport by M-cells, potassium transport, muscle contraction, and thyroid hormone synthesis. Immunohistochemical staining confirmed the absence of CA VI in the submandibular gland of Car6 (-/-) mice. Immunostaining of the trachea and lung samples revealed no differences between the knockout and wild type groups nor were any morphological changes observed. The present findings can help us to recognize novel functions for CA VI-one of the major protein constituents of saliva and milk.