Gas Chromatography-Mass Spectrometry (GC-MS) Metabolite Profiling of Citrus limon (L.) Osbeck Juice Extract Evaluated for its Antimicrobial Activity Against Streptococcus mutans.

Purpose The present study aimed to determine the antimicrobial nature of Citrus limon juice extract against Streptococcus mutans and to identify its metabolic profile by gas chromatography-mass spectrometry (GC-MS) technique. Materials and methods The cariogenic bacteria S. mutans were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India, and revived on brain heart infusion (BHI) agar. The C. limon (L.) Osbeck fruits were authenticated from the University of Trans-Disciplinary Health Sciences and Technology (TDU), Bengaluru, India. The antibacterial property of lyophilized lemon juice extract (LJE) dissolved in methanol was evaluated against S. mutans (MTCC 497) by the agar well diffusion assay. GC-MS technique was employed to identify the volatile metabolite profile of the methanolic LJE sample. The metabolite masses of the respective compounds were identified using the National Institute of Standards and Technology (NIST) library. Results The methanolic LJE sample concentration from 5 to 25 mg/ml did not demonstrate antimicrobial activity, while 30 to 100 mg/ml displayed antibacterial activity against S. mutans. Chlorohexidine (100 µg/ml) was used as the positive control, while methanol was used as the negative control. Significant antimicrobial metabolites were detected in the methanolic LJE sample by GC-MS analysis. Maleic anhydride, succinic anhydride, 6-Oxa-bicyclo[3.1.0] hexan-3-one, and 3-methyl-2,5-Furandione were the key metabolites identified in the methanolic LJE sample.  Conclusion The present study reports that C. limon is a potential candidate for the pharmaceutical industry as it possesses bioactive compounds demonstrating therapeutic properties. Further investigations are warranted to determine the individual and synergetic effects of identified metabolites in the methanolic LJE sample for its antimicrobial property. Special attention needs to be focussed on translational research for the development of anti-caries products from C. limon.

Production of antipeptide antibodies.

Peptides (8-20 residues) are as effective as proteins in raising antibodies, both polyclonal and monoclonal with a titer above 20,000 easily achievable. A successful antipeptide antibody production depends on several factors such as peptide sequence selection, peptide synthesis, peptide-carrier protein conjugation, the choice of the host animal, and antibody purification. Peptide sequence selection is likely the most difficult and critical step in the development of antipeptide antibodies. Although the format for designing peptide antigens is not precise, several guidelines can help maximize the likelihood of producing high-quality antipeptide antibodies. Typically, 5-20 mg of peptide is enough for raising an antibody, for preparing a peptide affinity column, and for antibody titer determination using an enzyme-linked immunosorbent assay (ELISA). Usually, it takes 3 months to raise a polyclonal antipeptide antibody from a rabbit that yields ~90 mL of serum which translates into approximately 8-10 mg of the specific antibody after peptide affinity purification.

Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation.

To utilize biologic mechanisms to elicit controlled release in response to disease, protease-sensitive devices have been created. Hydrogels were created with pendant peptide-drug complexes. For the matrix metalloproteases (MMPs) examined, a length of six amino acids greatly improved the specificity of the peptide (k(cat)/K(m) approximately 2.4+/-0.1x10(4)M(-1)s(-1)) over shorter sequences (k(cat)/K(m) approximately 4.4+/-0.2x10(2)M(-1)s(-1)). The peptides did not exhibit anti-proliferative effects upon cancer cells, and peptide-platinum complexes showed similar anti-proliferative effects upon the cancer cells compared to the free platinum drugs. Once the peptide-drug complex was incorporated into the hydrogels, the release was dependent upon the presence of MMP in the solution with approximately 35% of platinum released from hydrogels in the presence of MMP and only 10% without MMP in the week examined. The released drug exhibited the expected anti-proliferative activity over several days of incubation. The MMP selective drug delivery holds much potential for treatment of cancer and other diseases.

Three-dimensional chemical structures by protein functionalized micron-sized beads bound to polylysine-coated silicone surfaces.

A novel method is described here that allows three-dimensional (3D) control of both chemistry and morphology by a series of wet chemical steps: the attachment of protein functionalized micron-sized beads onto a flat silicone surface that has been functionalized with a distinct chemical modification. Bovine serum albumin (BSA), laminin, or polylysine is covalently bound to 6.5-microm-diameter spherical beads. A chemical method is then used to bind these beads to a flat silicone surface that is subsequently functionalized with polylysine. This process leads to a nonspecific cell adhesive background on the flat surface (polylysine) with the option of differing chemistry on the third-dimension due to the protein BSA or laminin on the bead protruding from the surface. The beads do not detach during cyclic stretching in vitro. Neo-natal rat cardiac fibroblasts are cultured on the beaded surfaces and compared with fibroblasts cultured on nonbeaded, flat polylysine surfaces. Fibroblast plating density, integrin, and physical responses are examined as a function of varying the ligands on the beads.

RGD and YIGSR synthetic peptides facilitate cellular adhesion identical to that of laminin and fibronectin but alter the physiology of neonatal cardiac myocytes.

In the mammalian heart, the extracellular matrix plays an important role in regulating cell behavior and adaptation to mechanical stress. In cell culture, a significant number of cells detach in response to mechanical stimulation, limiting the scope of such studies. We describe a method to adhere the synthetic peptides RGD (fibronectin) and YIGSR (laminin) onto silicone for culturing primary cardiac cells and studying responses to mechanical stimulation. We first examined cardiac cells on stationary surfaces and observed the same degree of cellular adhesion to the synthetic peptides as their respective native proteins. However, the number of striated myocytes on the peptide surfaces was significantly reduced. Focal adhesion kinase (FAK) protein was reduced by 50% in cardiac cells cultured on YIGSR peptide compared with laminin, even though beta(1)-integrin was unchanged. Connexin43 phosphorylation increased in cells adhered to RGD and YIGSR peptides. We then subjected the cardiac cells to cyclic strain at 20% maximum strain (1 Hz) for 48 h. After this period, cell attachment on laminin was reduced to approximately 50% compared with the unstretched condition. However, in cells cultured on the synthetic peptides, there was no significant difference in cell adherence after stretch. On YIGSR peptide, myosin protein was decreased by 50% after mechanical stimulation. However, total myosin was unchanged in cells stretched on laminin. These results suggest that RGD and YIGSR peptides promote the same degree of cellular adhesion as their native proteins; however, they are unable to promote the signaling required for normal FAK expression and complete sarcomere formation in cardiac myocytes.

Catching protein antigens by antibody affinity electrophoresis.

A new kind of affinity electrophoresis called antibody affinity electrophoresis is a technique used to capture protein antigens based on their interactions with specific monoclonal or polyclonal antibodies incorporated in the polyacrylamide gel. Polyclonal anti-glutathione-S-transferase (anti-GST), monoclonal anti-bovine serum albumin (anti-BSA), and polyclonal anti-human alpha-lactalbumin are embedded in distinct areas of a 7.5% native polyacrylamide gel. Some of the embedded antibodies get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these antibodies do not show significant electrophoretic mobility, as compared to their specific protein antigen analytes. We observed that electrophoretic migration of GST, BSA, and protein G ceases when they encounter anti-GST, anti-BSA, and immunoglobulin G, respectively.

Derivatization of surface-bound peptides for mass spectrometric detection via threshold single photon ionization.

Chemical derivatization of peptides allows efficient F2 laser single photon ionization (SPI) of Fmoc-derivatized peptides covalently bound to surfaces. Laser desorption photoionization mass spectrometry using 337-nm pulses for desorption and 157.6-nm pulses for threshold SPI forms large ions identified as common peptide fragments bound to either Fmoc or the surface linker. Electronic structure calculations indicate the Fmoc label is behaving as an ionization tag for the entire peptide, lowering the ionization potential of the complex below the 7.87-eV photon energy. This method should allow detection of many molecular species covalently or electrostatically bound to surfaces.

GRGDSP peptide-bound silicone membranes withstand mechanical flexing in vitro and display enhanced fibroblast adhesion.

Mechanobiological studies of cardiac tissue require devices that allow forces to be exerted on cells in vitro. Silicone elastomer is often used in these devices because it is flexible and transparent, permitting optical imaging of the cells. However, native untreated silicone is hydrophobic and is unsuitable for cell culture. Peptides covalently bound to silicone surfaces are examined here for the enhancement of cellular adhesion during in vitro dynamic flexing. A procedure is described for the chemical modification of medical grade silicone membranes with covalently bound GRGDSP peptides. The conditions for mechanical studies of cardiac cell cultures are then duplicated and it is demonstrated that the peptide layers survive 48 h of mechanical flexing in vitro. Specifically, mechanical flexing in vitro of the 30 pmol/cm2 peptide-modified silicone membranes has no significant effect on the amount of peptides that remains bound to the surface. Cardiac fibroblasts display enhanced adhesion to these peptide-bound silicone membranes for at least 24 h of growth, compared with native silicone or tissue culture polystyrene. The effects of serum versus serum-free media on fibroblast growth are also examined.