Characterization of Mouse Tissue Kallikrein 5

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (KIk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling

Preliminary investigations on the serine and aspartic protease inhibitors from <i>Nothopegia</i> <i>beddomei</i>

Aqueous stem bark extract of Nothopegia beddomei was assayed for serine and aspartic protease inhibitors by using trypsin and pepsin as the target enzymes, respectively. Crude bark extract was dialyzed and subjected to inhibition assays. The thermal stabilities of serine and aspartic protease inhibitors were studied by incubating the extract at 4 ºC, room temperature and 37 ºC for a period of one month and subjecting to higher temperatures (55 ºC, 75 ºC and 95 ºC) for 15 minutes. The crude bark extract of N. beddomei exhibited serine and aspartic protease inhibitory activities. However, the serine protease inhibitory activity was significantly higher. The approximate molecular mass of both serine and aspartic protease inhibitors were more than 8 kDa. Both types of inhibitors were identified as mixtures of thermally stable and labile compounds due to their variations in inhibitory activities with time at different temperatures. Attempts made to purify the protease inhibitors using ion exchange chromatography and ammonium sulphate precipitation were unsuccessful. In conclusion, the results indicate both serine and aspartic protease inhibition activities in N. beddomei bark extract are governed by mixtures of protenaceous and non-protenaceous molecules

Characterization of Luteinizing Hormone and Luteinizing Hormone Receptor and Their Indispensable Role in the Ovulatory Process of the Medaka

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare's serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation

Biochemical characterization of human kallikrein 8 and its possible involvement in the degradation of extracellular matrix proteins

Human kallikrein 8 (KLK8) is a member of the human kallikrein gene family of serine proteases, and its protein, hK8, has recently been suggested to serve as a new ovarian cancer marker. To gain insights into the physiological role of hK8, the active recombinant enzyme was obtained in a pure state for biochemical and enzymatic characterizations. hK8 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type IV, fibrinogen, and high-molecular-weight kininogen. hK8 also converted human single-chain tissue-type plasminogen activator (65 kDa) to its two-chain form (32 and 33 kDa) by specifically cleaving the peptide bond Arg275–Ile276. This conversion resulted in a drastic increase in the activity of the activator toward the fluorogenic substrate Pyr-Gly-Arg-MCA and plasminogen in the absence of fibrin. Our findings suggest that hK8 may be implicated in ECM protein degradation in the area surrounding hK8-producing cells

Expression of cyclooxygenase-2 and prostaglandin receptor EP4b mRNA in the ovary of the medaka fish, Oryzias latipes: Possible involvement in ovulation

In vitro ovulation of mature medaka ovarian follicles was inhibited by inhibitors of cyclooxygenase (COX) or by an antagonist of the prostaglandin E2 receptor (EP). Of the three medaka COX genes, ptgs2 was most dominantly expressed in the fish ovary. The ptgs2 transcript was detected in all sizes of growing follicles. In a 24-h spawning cycle, large-sized follicles contained ptgs2 mRNA at a fairly constant level. The levels of COX enzyme activity and prostaglandin E2 were also constant in the large-sized follicles during the spawning cycle. The expression of prostaglandin E2 receptor EP4b (ptger4b) mRNA was drastically upregulated in the large-sized follicles as the ovulation time approached. The current results indicate that prostaglandin E2. which might be produced by COX-2, is involved in the ovulation of medaka, and that EP4b is likely the receptor responsible for exerting the action of prostaglandin E2 in the process

Morphological, antimicrobial, durability, and physical properties of untreated and treated textiles using silver-nanoparticles

Silver nanoparticles are often applied to textiles for their strong antimicrobial activity and potential uses in various applications. The treatment of fabrics with silver nanoparticles has often involved complex or expensive processes, required surface post treatment, lacked durability and altered desirable properties related to the comfort of the fabric. In this paper, a systematic study has been performed to identify a simple yet durable and economical approach to apply silver nanoparticles on cotton fabrics with minimum alterations to the fabrics' physical properties. An ex situ chemical and in situ photo reduction approaches of silver-nanoparticle treatment on cotton fabrics were investigated, comparing the morphology, antimicrobial, durability of the treatment after wash and physical properties. Results indicate that the in situ approach was favorable toward aforementioned requirements and could retain its properties close to the original fabric. Methodology presented here to study effects of ex situ and in situ treatments of silver nanoparticles on textiles could be of interest to other applications.</p