Bioactive Peptides from Algae: Traditional and Novel Generation Strategies,Structure-Function Relationships,and Bioinformatics as Predictive Tools for Bioactivity

Over the last decade, algae have been explored as alternative and sustainable protein sources for a balanced diet and more recently, as a potential source of algal-derived bioactive peptides with potential health benefits. This review will focus on the emerging processes for the generation and isolation of bioactive peptides or cryptides from algae, including: (1) pre-treatments of algae for the extraction of protein by physical and biochemical methods; and (2) methods for the generation of bioactive including enzymatic hydrolysis and other emerging methods. To date, the main biological properties of the peptides identified from algae, including anti-hypertensive, antioxidant and anti-proliferative/cytotoxic effects (for this review, anti-proliferative/cytotoxic will be referred to by the term anti-cancer), assayed in vitro and/or in vivo, will also be summarized emphasizing the structure–function relationship and mechanism of action of these peptides. Moreover, the use of in silico methods, such as quantitative structural activity relationships (QSAR) and molecular docking for the identification of specific peptides of bioactive interest from hydrolysates will be described in detail together with the main challenges and opportunities to exploit algae as a source of bioactive peptides.     

Modulation of nicotinamide adenine dinucleotide phosphate oxidase activity through sequential posttranslational modifications of p22 phagocytic oxidase during capacitation and acrosome reaction in goat spermatozoa

Superoxide anion radical, produced in low quantities, plays a positive role in sperm function. Spermatozoa produce superoxide anion radical during posttesticular development, which shows an abrupt increase during capacitation. The NAD phosphate oxidase (NOX) family members NOX2 and NOX5 are the 2 enzymes implicated in superoxide production in spermatozoa. We examined the organization of NOX2 in goat spermatozoa during epididymal maturation, capacitation, and acrosome reaction. Spermatozoa from testis, caput epididymidis, corpus epididymidis, and cauda epididymidis possessed components of the phagocytic oxidase (PHOX; i.e., gp91phox, p22phox, p67phox, p47phox, p40phox), and ras-related C3 botulinum toxin substrate 1/2 (Rac1/2) on spermatozoa, and their concentrations did not show significant alterations during epididymal maturation. During capacitation in vitro, p22phox underwent Thr-phosphorylation, which resulted in a mobility shift of the corresponding band toward greater molecular mass. The Rac1/2 also showed a mobility shift from 32 to 23 kDa during capacitation. During progesterone-induced acrosome reaction, the spermatozoa experienced a total loss of p22phox and p47phox. The p47phox, but not p22phox, was detected in the exocytic vesicles of the acrosome. The Thr-phosphorylated form of p22phox was ubiquitinated and degraded through proteasome-mediated pathways in goat sperm cell lysates. Thus, Thr phosphorylation of p22phox acts as a regulatory switch in goat spermatozoa that transiently activates the NOX2 system during capacitation and subsequently directs it for degradation through the ubiiquitin-proteasomal pathway during progesterone-induced acrosome reaction.     

Pharmacokinetics of cefpodoxime in plasma and subcutaneous fluid following oral administration of cefpodoxime proxetil in male beagle dogs

Kumar, V., Madabushi, R., Lucchesi, M. B. B., Derendorf, H. Pharmacokinetics of cefpodoxime in plasma and subcutaneous fluid following oral administration of cefpodoxime proxetil in male beagle dogs. J. vet. Pharmacol. Therap. 34 , 130–135. Pharmacokinetics of cefpodoxime in plasma (total concentration) and subcutaneous fluid (free concentration using microdialysis) was investigated in dogs following single oral administration of prodrug cefpodoxime proxetil (equivalent to 5 and 10 mg/kg of cefpodoxime). In a cross over study design, six dogs per dose were utilized after a 1 week washout period. Plasma, microdialysate, and urine samples were collected upto 24 h and analyzed using high performance liquid chromatography. The average maximum concentration (C_max) of cefpodoxime in plasma was 13.66 (±6.30) and 27.14 (±4.56) μg/mL with elimination half‐life (t_1/2) of 3.01 (±0.49) and 4.72 (±1.46) h following 5 and 10 mg/kg dose, respectively. The respective average area under the curve (AUC_0–∞) was 82.94 (±30.17) and 107.71 (±30.79) μg·h/mL. Cefpodoxime was readily distributed to skin and average free C_max in subcutaneous fluid was 1.70 (±0.55) and 3.06 (±0.93) μg/mL at the two doses. Urinary excretion (unchanged cefpodoxime) was the major elimination route. Comparison of subcutaneous fluid concentrations using pharmacokinetic/pharmacodynamic indices of fT_>MIC indicated that at 10 mg/kg dose; cefpodoxime would yield good therapeutic outcome in skin infections for bacteria with MIC₅₀ upto 0.5 μg/mL while higher doses (or more frequent dosing) may be needed for bacteria with higher MICs. High urine concentrations suggested cefpodoxime use for urinary infections in dogs.     

Cytokine profiles, apoptosis and pathology of experimental Pasteurella multocida serotype A1 infection in mice

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P < 0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections.     

Sonographic characteristics of goat testis on water bath based ultrasonography

Assessing the health of the testes in domestic animals is an important aspect of the breeding soundness examination and selection. The aim of the present study was to develop a simple method for scanning and to establish ultrasonographically the gross anatomic structures of the goat testes. Six adult male goats were examined to study the sonographic appearance of normal testes and epididymides using a water bath based ultrasound scanning technique. The ultrasonographic examinations were done using a 5–9 MHz/60 mm (7.5 MHz) linear-array transducer and a B-mode scanner. The ultrasonographic examination was performed in goats after standardizing the procedure on six testes collected from slaughter house. Results showed that in live goats when the probe was placed directly over the scrotum it gave distorted and unclear image. In water bath method the entire scrotum was dipped into a container filled with water and linear probe was used to observe the sonographic features of the testis. Each testis was viewed vertically, resulting in longitudinal image which was frozen, measured and printed through a thermal printer. The results of the ultrasonogram revealed that the testicular parenchyma was homogenous and moderately echogenic throughout. The diameter (mean±se) of the right and left testes was 4.47±0.14 and 4.42±0.07 cm respectively and no significant difference was observed between the testes. The mediastinum testis was a 1.50±0.22 cm wide linear structure of greater echogenicity than the testicular parenchyma when viewed in the transverse plane and nearly circular echogenic “spot” in the midline of the testis when viewed horizontally. The head and tail of the epididymides were easily identified on all the testes, but the epididymal body and ductus deferens were difficult to identify consistently. The tail of the epididymis was easily identified on the distal end of the testis with sonolucent tubules and appeared sonographically as a ‘peaked cap’ upon the testicular parenchyma. The diameter (mean±se) of the tail of right and left epididymis was 2.11±0.18 and 1.92±0.06 cm and no significant difference was observed between epididymides. The vascular pampiniform plexus (1.42±0.18 cm) was easily identified on the proximal end of the testes. The tunics of the testes appeared as a bright echogenic line. Inter-testicular septum appeared between testes as a hyperechoic line. It is concluded that ultrasonography permits a noninvasive evaluation of the internal structure of the scrotum and testes and water bath based sonographic examination may prove to be a valuable simple diagnostic methodology for evaluating physiopathologic conditions of goat testes and can be employed as a routine investigative method during breeding soundness and clinical examination.     

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n = 94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes 460, 1044, 657 and 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.     

Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo

Adiponectin is an adipocyte‐derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p < .05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in‐vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10 µg/ml alone or with follicle stimulating hormone (FSH) at 30 ng/ml) or Insulin‐like growth factor I (IGF‐I) at 10 ng/ml for 48 hr after obtaining 75%–80%s confluency. Adiponectin at 10 µg/ml increased IGF‐I‐induced estradiol (E₂) and progesterone (P₄) secretion and FSH‐induced E₂ secretion from GC and also increased the abundance of factors involved in E₂ and P₄ production (cytochrome P45019A1 [CYP19A1] and 3‐beta‐hydroxysteroid dehydrogenase [3β‐HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.     

Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis

A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation can be derived that allows demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).     

Studies on immunocompetence status in two turkey varieties in India

(1) Two hundred and twenty-seven adult turkeys of both sexes, of two varieties (104 Black and 123 White) were used to evaluate their immunocompetence status and body weights. (2) Response to sheep red blood cells (SRBC) (humoral immunity) was measured by Haemagglutination (HA) test 5 days post immunisation (dpi) and expressed as log2 values. Mercaptoethanol resistant (MER) antibodies representing IgG were determined by Mercaptoethanol HA test and Mercaptoethanol sensitive (MES) antibodies, representing IgM as the difference in total HA titre and IgG. Serum lysozyme concentrations were estimated by 'Lysoplate assay' and expressed in log2 values. (3) Least squares analysis of variance revealed that the White variety had higher adult body weight (4.788 +/- 0.040 kg) than the Black (3.774 +/- 0.044 kg). Sexual dimorphism was apparent and meals were heavier than females in both varieties. The interaction effect of variety and sex on body weight was also significant. (4) Least squares means for immunological traits, namely, total anti-SRBC antibodies, MER, MES titres and serum lysozyme were 7.161 +/- 0.189, 0.801 +/- 0.071, 6.362 +/- 0.160 and 1.766 +/- 0.043 microg/ml, respectively. The Black variety had a higher MES antibody titre than the White. (5) Sex had an effect on all the immunological traits except on MER titres. Females generally had higher anti-SRBC, MER and MES titres and serum lysozyme. The variety x sex interaction effect was significant for MES titres and serum lysozyme. White males had the lowest MES titres.