Inhibition effect of rosette formation between human lymphocytes and sheep erythrocytes by specific heptapeptide isolated from uremic fluid and its analogs.

One of middle molecular substances (H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH) and its two analogs, in which the proline residue in position 2 was replaced by glycine and the alanine residue in position 3 was replaced by valine exert a inhibition effect on $\text{in vitro}$ E-rosette formation. Its synthetic two analogs showed diminished biological activity compared to native heptapeptide.     

Identification and synthesis of a tripeptide in ECUM fluid of an upemic patient

An unidentified ninhydrin and Pauly reaction positive substance of basic nature was found in the ECUM fluid of an uremic patient. This substance was isolated from ECUM fluid by the methods of ultrafiltration method and gel-filtration, and identified as H-His-Gly-Lys-OH by amino acid analysis, manual Edman degradation method and physical constants and analytical data of synthetic tripeptide.     

Isolation, structure and synthesis of a heptapeptide with in vitro ACTH-releasing activity from porcine hypothalamus

Significant CRF activity was found in a fraction with Rf = 0.82-0.7 or VE/VT = 0.41-0.48 obtained by gel filtration of acid extracts of pig hypothalami on Sephadex G-25. The activity of this fraction decreased markedly during subsequent purification, particularly in the last two steps. From this fraction, a heptapeptide with significant ACTH releasing activity in vitro, was isolated in pure state, and its amino acid sequence was established as H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH. This heptapeptide was synthesized by solid phase methods. The CRF activity of synthetic heptapeptide in vitro was low but could be potentiated by a cofactor fraction from rat hypothalamic extract.     

Identification of a tumor inhibitory factor in rat ascites fluid

A polypeptide which inhibits the growth of human carcinoma cells has been characterized from Novikoff rat ascites fluid. This tumor inhibitory factor co-purified with transforming growth factor activity through acid/ethanol extraction and Bio-Gel chromatography. The two activities were completely separated by reverse phase HPLC. The tumor inhibitory factor is heat stable and requires disulfide bonds for bioactivity. This factor inhibited the anchorage independent growth of the more differentiated human colon carcinoma cell lines but did not affect the less differentiated carcinoma cells. The presence of stimulatory and inhibitory activities in the same extracts suggests that the relative concentrations of these factors may be important in the control of cell growth.     

Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy

A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.     

Compensatory and antiamnestic effects of heptapeptide Selank in monkeys

The work presents results of study of role of heptapeptide Selank—an anxiolytic from group of biologically active peptides—in compensation of disturbed psychic and homeostatic functions in monkeys. New data have been obtained which indicate that an intranasal administration of Selank produces long-term changes of the monkey behavior disturbed during neurosis: elimination of fear and aggression and an increase of explorative activity as well as facilitation of handling reactions and communicational relations. It has been established that on the background of the Selank there occurs a long compensation of disturbed psychic functions (processes of memory) and of homeostatic parameters. It has been shown that unlike the earlier studied neurohormones (thyroliberin and ACTH_4?10), the antistress Selank effects do not depend on the type of neurotic disturbances and have long-term compensatory character. Comparison of the data obtained on monkeys with results of similar studies on the more low-organized mammals (rodents) allow suggesting that the new peptide preparation Selank is a promising agent for correction of various psychoemotional disturbances (alarm-and depression-like disorders).     

Isolation, structure and biological activity of the Trp-containing pentapeptide from uremic fluid.

Trp-containing pentapeptide was isolated from uremic fluid of an uremic patient by ultrafiltration with Amicon membranes followed by gel filtrations. The peptide thus obtained was identified as H-Asp-Leu-Trp-Gln-Lys-OH by amino acid analysis, manual Edman degradation method, physical constants and analytical data of synthetic pentapeptide. Structural similarity was soon realized between this peptide and pentapeptide moiety corresponding to position 123 through 127 of β-chain of fibrinogen. E-rosettes inhibition test was shown this pentapeptide to have an inhibition activity by amount more than l.Omg/ml.     

An actin-interacting heptapeptide in the cofilin sequence

Cofilin, a 21-kDa actin-binding protein, has a hexapeptide sequence DAIKKK which is identical to the N-terminal portion (residues 2-7) of tropomyosin. The synthetic heptapeptide, DAIKKKL, corresponding to residues 122-128 of cofilin, inhibited the binding of cofilin to F-actin in a dose-dependent manner. The heptapeptide cosedimented with F-actin, decreased the fluorescence intensity of pyrene-labeled F-actin, and increased the rate of polymerization of G-actin. The hexapeptides, DIKKKL and DAIKKL, also inhibited the binding of cofilin to F-actin and affected the fluorescence intensity of pyrene-labeled F-actin and the rate of actin polymerization, like the heptapeptide. However, their effects were weaker than those of the heptapeptide. Moreover, the pentapeptide, DIKKL, had little or no effect. These results suggest that the heptapeptide sequence is specific for the interaction with actin and, therefore, may constitute part of the actin-binding domain of cofilin.     

Identification and synthesis of a unique thiocarbazate cathepsin L inhibitor

Library samples containing 2,5-disubstituted oxadiazoles were identified as potent hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) directed at discovering inhibitors of cathepsin L. However, when synthesized in pure form, the putative actives were found to be devoid of biological activity. Analyses by LC-MS of original library samples indicated the presence of a number of impurities, in addition to the oxadiazoles. Synthesis and bioassay of the probable impurities led to the identification of a thiocarbazate that likely originated via ring opening of the oxadiazole. Previously unknown, thiocarbazates (-)-11 and (-)-12 were independently synthesized as single enantiomers and found to inhibit cathepsin L in the low nanomolar range.     

Identification and function of proteolysis regulators in seminal fluid

Proteins in the seminal fluid of animals with internal fertilization effect numerous responses in mated females that impact both male and female fertility. Among these proteins is the highly represented class of proteolysis regulators (proteases and their inhibitors). Though proteolysis regulators have now been identified in the seminal fluid of all animals in which proteomic studies of the seminal fluid have been conducted (as well as several other species in which they have not), a unified understanding of the importance of proteolysis to male fertilization success and other reproductive processes has not yet been achieved. In this review, we provide an overview of the identification of proteolysis regulators in the seminal fluid of humans and Drosophila melanogaster, the two species with the most comprehensively known seminal fluid proteomes. We also highlight reports demonstrating the functional significance of specific proteolysis regulators in reproductive and post‐mating processes. Finally, we make broad suggestions for the direction of future research into the roles of both active seminal fluid proteolysis regulators and their inactive homologs, another significant class of seminal fluid proteins. We hope that this review aids researchers in pursuing a coordinated study of the functional significance of proteolysis regulators in semen. Mol. Reprod. Dev. © 2012 Wiley Periodicals, Inc.     

Evidence for a novel FMRFamide-related heptapeptide in the pulmonate snail Siphonaria pectinata

Extracts of whole false limpets (Siphonaria pectinata) were analysed to determine their complement of FMRFamide-related peptides. As in other pulmonates, FMRFamide itself was found to account for only a portion of the immunoreactivity; the largest immunoreactive peptide peak eluted during HPLC under acidic conditions at the same position as a peak also found in other pulmonates. This major peak was resolved into two components by HPLC at neutral pH, and one component was identified as the heptapeptide amide, GDPFLRFamide, previously described from Lymnaea. The amino acid composition of the second component indicates that it is also a heptapeptide, but that it has two Asx (aspartic acid or asparaginyl) residues instead of the one found in the previously identified pulmonate heptapeptides.     

Identification of a unique idiotype in cerebrospinal fluid and serum of a patient with multiple sclerosis

Anti-idiotypic antibodies were prepared in mouse ascites fluid against the CSF-IgG of a patient with multiple sclerosis. After adsorption with pooled human IgG, the ascites fluid antibodies precipitated 20% of labeled autologous CSF IgG. By using a competitive radioimmunoassay, less than one microgram of unlabeled CSF IgG produced 50% inhibition of binding autologous 125I-labeled CSF IgG, whereas 50 micrograms of normal HIgG was not inhibitory. The idiotype could be found in both serum and CSF IgG and persisted over a 5-year period. The absolute concentration of idiotype in the CSF varied somewhat but remained from 4 to 10 times greater than that of the serum. One of 14 heterologous MS CSF was found to contain small amounts of inhibitory protein; eight CSF from patients with other neurologic diseases did not contain the idiotype.     

Identification of glycoproteins in human cerebrospinal fluid with a complementary proteomic approach

Biomarkers are pressingly needed to assist with the clinical diagnosis of neurodegenerative diseases and/or the monitoring of disease progression. Glycoproteins are enriched in bodily fluids such as human cerebrospinal fluid (CSF), an ideal source for discovering biomarkers due to its proximity to the central nervous system (CNS), and consequently can serve as diagnostic and/or therapeutic markers for CNS diseases. We report here an in-depth identification of glycoproteins in human CSF using a complementary proteomic approach which integrated hydrazide chemistry and lectin affinity column for glycoprotein enrichment, followed by multidimensional chromatography separation and tandem mass spectrometric analysis. Using stringent criteria, a total of 216 glycoproteins, including many low-abundance proteins, was identified with high confidence. Approximately one-third of these proteins was already known to be relevant to the CNS structurally or functionally. This investigation, for the first time, not only categorized many glycoproteins in human CSF but also expanded the existing overall CSF protein database.     

Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis.

Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the cell surface that is used among all eukaryotes. A common core structure, EthN-P-Man3-GlcN-PI, is synthesized by sequential transfer of sugars and ethanolamine-P to PI and is highly conserved between organisms. We have screened for natural compounds that inhibit GPI-anchoring in yeast and have identified a terpenoid lactone, YW3548, that specifically blocks the addition of the third mannose to the intermediate structure Man2-GlcN-acyIPI. Consistent with the block in GPI synthesis, YW3548 prevents the incorporation of [3H]myo-inositol into proteins, transport of GPI-anchored proteins to the Golgi and is toxic. The compound inhibits the same step of GPI synthesis in mammalian cells, but has no significant activity in protozoa. These results suggest that despite the conserved core structure, the GPI biosynthetic machinery may be different enough between mammalian and protozoa to represent a target for anti-protozoan chemotherapy.     

Identification of a methyltransferase from Mycobacterium smegmatis involved in glycopeptidolipid synthesis

Glycopeptidolipids (GPLs) are major components of the cell walls of several species of mycobacteria. We have isolated a transposon mutant of Mycobacterium smegmatis that is unable to synthesize mature GPLs and that displays a rough colony morphology. The disrupted gene, mtf1, shares a high degree of homology with several S-adenosylmethionine-dependent methyltransferases. The enzyme encoded by mtf1 is required for the methylation of a single rhamnose residue that forms part of the conserved GPL core structure. This conclusion is supported by the finding that (a) the mutant synthesized only GPLs with undermethylated (either mono- or nonmethylated instead of di- or trimethylated) rhamnose residues; (b) complementation of the mutant with a wild-type copy of mtf1 restored high levels of synthesis of GPLs containing di- and trimethylated rhamnose; and (c) S-adenosylmethionine-dependent methylation of rhamnosylated GPLs could be detected in cell lysates of wild-type cells and mtf1-complemented mutant cells, but not in mutant cells lacking intact mtf1. Structural analysis of wild-type and mutant GPLs suggests that disruption of mtf1 specifically inhibits addition of O-methyl groups to the 3 (or 2)-position of the rhamnose. In the absence of 3-O-methylation, further methylation of GPL rhamnose is apparently inhibited, and overall GPL synthesis is down-regulated by 90%.     

Identification and synthesis of a trisaccharide produced from lactose by transgalactosylation

Enzymatic transgalactosylation of lactose by means of Streptococcus thermophilus, subspecies DN-001065, led to a mixture of D-galactose (approximately 4%), D-glucose (approximately 15%), lactose (approximately 51%), minor disaccharides (6%), trisaccharides (approximately 20%) and tetrasaccharides (3%). The major trisaccharide (approximately 16%) was identified by NMR spectroscopy and chemical synthesis as being the known beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->4)-D-glucos e (3'-beta-D-galactopyranosyl-lactose). It was purified from a mixture of peracetylated oligosaccharides by column chromatography followed by deacetylation. For the first time, 3'-beta-D-galactopyranosyl-lactose has been obtained on the 1 g scale, by resorting to simple techniques and equipment. NMR spectra have been unambiguously assigned.     

In vitro and in vivo TNFalpha synthesis modulation by methylguanidine, an uremic catabolyte.

This study was performed in order to examine whether the uraemic toxin, methylguanidine (MG), can modulate tumor necrosis factor alpha (TNF alpha) release by activated macrophages. In this study we have evaluated the ability of MG to influence TNF alpha release in vitro, in Escherichia coli lypopolysaccharide- (LPS)-stimulated J774 cells preincubated overnight with MG, and in vivo in rats treated with MG before and after LPS challenge. Parallel experiments employing N(G)-nitro-L-arginine methyl esther (L-NAME) were also carried out for comparison. The effect of LPS (6 x 10(3) u/ml) on TNF alpha release by J774, following overnight incubation with MG or L-NAME (1 mM), was examined 3 hours after LPS challenge. LPS-stimulated J774 released 287.83+/-88 u/ml TNF alpha into the culture medium. MG (1 mM) significantly inhibited TNF alpha release by 73% (P<0.05). L-NAME (1 mM) significantly inhibited TNF alpha release too by 72.88% (P<0.05). The effect of MG and L-NAME have been also studied in vivo. Serum TNF alpha levels in LPS treated rats 2 h after LPS challenge were 88.33+/-31.7 u/ml as compared to the serum TNF alpha levels of control rats (undetectable). Treatment of rats with MG (30 mg/kg, i.p.) strongly and significantly reduced TNF alpha release (98.71% inhibition; with P<0.001); in the same experimental setting L-NAME (10 mg/kg, i.p.) also significantly reduced TNF alpha serum levels (76.47% inhibition; with P<0.01). These results could indicate that immune disfunction related to uremia may be related to the inhibitory capability of uremic catabolyte, MG, on TNF alpha synthesis and release.