Effect of incident light wavelength and corneal edema on light scattering and penetration: laboratory study of human corneas.

PURPOSE: The outcome of ultrashort pulse laser surgery of the cornea is strongly influenced by the light scattering properties of the tissue, for which little data are available. The purpose of the present study is to provide quantitative values for light scattering and its relation to the degree of edema. METHODS: An experimental optical measuring setup based on confocal geometry was used to measure the unscattered and scattered fractions of light transmitted by eye bank corneas presenting various degrees of edema. From these measurements, the effective light penetration depth in the cornea was calculated as a function of wavelength. RESULTS: Corneal transparency depends on the pathological state of the cornea and on wavelength. It may be predicted as a function of corneal thickness, ie, the degree of edema. In healthy and edematous cornea, the percentage of scattered light decreases with increasing wavelength. The total penetration depths at the wavelengths of ~1050 nm (which is used in typical clinical systems) and 1650 nm (which is recommended for future devices) are comparable; however, the former is limited by scattering, which degrades the laser beam quality, whereas the latter is only limited by optical absorption, which may be compensated for. CONCLUSIONS: The use of longer wavelengths should help improve the surgical outcome in ultrashort pulse laser surgery of the cornea when working on pathological tissue. A wavelength of approximately 1650 nm appears to be a good compromise, as it allows for reduced light scattering while keeping optical absorption reasonably low.

Antimicrobial activity of lipophilic avian eggshell surface extracts.

The avian eggshell cuticle is the waxy outermost layer of the mineralized eggshell in direct contact with the environment. In this study, lipophilic eggshell surface extracts from three domestic species were evaluated for their antimicrobial activity. Chicken and goose extracts demonstrated potent bactericidal activity against both Gram-positive and Gram-negative bacteria, while activity could not be detected for duck eggshell surface extracts. Using the chicken as a model species, evaluation of albumen, fecal material, and uropygial gland extracts eliminated these as a potential source of the observed activity. Results suggest that lipophilic components are incorporated into the egg during its formation and play a role in antimicrobial defense. This study represents the first successful extraction and evaluation of lipophilic antimicrobial components from the avian egg.

Antimicrobial effects of H4-(86-100), histogranin and related compounds--possible involvement of DNA gyrase.

Histone-derived antimicrobial peptides have been identified in various organisms from plants to humans. The rat histone H4 mRNA variants, H4-v.1 and rat histogranin (HNr) mRNAs, were recently reported to be involved in the synthesis of H4-(86-100) and its related peptide HNr, respectively. Herein, the two peptides were investigated for putative antimicrobial activity and found to inhibit growth of gram-negative (Escherichia coli, Pseudomonas aeruginosa) and gram-positive (Bacillus subtilis, Staphylococcus aureus) bacteria. Their inhibitory potencies in E. coli (LD(50): 3.48 and 4.34 microg x mL(-1)) are comparable to that of the antimicrobial peptide LL-37 (LD(50): 4.10 microg x mL(-1)). The antimicrobial activities of H4-(86-100) and HNr depend upon the integrity of the molecules, as precursors [H4-(84-102), pro-HNr] and fragments [bovine histogranin (HNb)-(1-13), HNb-(3-13), H4-(89-102) or OGP] are at least five times less potent than the parent peptides. Among various HN-like compounds, cyclo-(-Gly-pCl-Phe-Tyr-D-Arg) (compound 3) and N-5-guanidino pentanamide-(2R)-yl-2-N-(p-hydroxyphenylacetyl)-4-(p-chlorobenzoyl)-phenylene diamine (compound 8) display antimicrobial activities comparable to that of HNr. Interestingly, the antimicrobial activities of H4-(86-100), HNr and compound 3, like those of quinolone antibiotics acting as DNA gyrase poisons, are potentiated by ATP (1 mM) and coumermycin A1 (a DNA gyrase-linked ATPase inhibitor) and blocked by 2,4-dinitrophenol (DNP, an uncoupler of oxidative phosphorylation) and fluoroacetic acid (a metabolic poison). Finally, in vitro experiments indicate that H4-(86-100), HNr, compound 3 and compound 8, but not HNb-(1-13) or HNb-(3-13), inhibit DNA gyrase-mediated supercoiling of pBR322 DNA. These data indicate that the naturally occurring H4-(86-100) and HNr display antimicrobial effects that involve a modulation of ATP-dependent DNA gyrase.

Characterization, localization and possible anti-inflammatory function of rat histone H4 mRNA variants.

Two histone H4 mRNA variants, H4-v.1 and histogranin mRNAs, were detected in the rat genome and measured in various tissues and isolated alveolar macrophages. Medium to high levels of both mRNAs were present in the liver, adrenal glands, thymus, bone marrow and alveolar macrophages. H4-v.1 cDNA contained an open reading frame that coded for unmodified whole histone H4, whereas histogranin cDNA lacked the first ATG codon and contained an open reading frame that coded for modified (Thr89) H4-(84-102). The two genes displayed a sequence homologous (> 80%) to the open reading frame of core H4 somatic (H4s) and H4 germinal (H4g) and their variant nature was supported by the absence of histone consensus palindromic and purine-rich sequences in the proximal 3'UTR, and the presence of a polyadenylation signal in the distal 3'UTR and of specific upstream transcription factor-binding sites. H4-v.1 and histogranin transcripts, but not H4s transcript, were selectively induced by lipopolysaccharide and/or interferon gamma in alveolar macrophages. In vitro transcription/translation experiments with H4-v.1 and histogranin cDNA pCMV constructs produced peptides with the molecular mass (2 kDa) of the alternative histone H4 translation product which, like synthetic H4-(86-100) and [Thr89]H4-(86-100) or rat histogranin, inhibited lipopolysaccharide-induced prostaglandin E(2) release from rat alveolar macrophages. The synthetic peptides also inhibited the secretion of the CXC chemokine interleukin-8 (GRO/CINC-1) in response to lipopolysaccharide. The presence of H4-v.1 and histogranin mRNAs in tissues wherein immune reactions take place and the inhibitory effects of their translation products on prostaglandin E(2) and interkeukin-8 secretion by activated alveolar macrophages suggest an anti-inflammatory function.

Correlation between the expression of the histone H4 mRNA variant H4-v.1 and the levels of histone H4-(86-100) and H4-(89-102) (OGP) in various rat tissues and alveolar macrophages.

We studied the expression of the osteogenic and antinociceptive C-terminal histone H4-related peptide fragments, H4-(89-102) (OGP) and H4-(86-100), respectively, within various rat tissues and isolated alveolar macrophages (AM) by radioimmunoassay (RIA). OGP was located mainly within the bone marrow, spleen, thymus, and lungs whereas H4-(86-100) was more concentrated within the bone marrow, lymph nodes, spinal cord, pituitaries and thymus. The expression pattern of the two peptides showed similarities with the tissue expression pattern of the histone H4 mRNA variant H4-v.1. In rat AM, OGP and H4-(86-100) levels were significantly stimulated (2.6- and 1.9-fold, respectively) by LPS (1 microg/ml), along with H4-v.1 mRNA (4.1-fold), but not whole histone H4 (1.1-fold) nor total histone H4 mRNA (1.1-fold). The results suggest that H4-v.1 mRNA may play a role in the synthesis of the naturally occurring peptides H4-(86-100) and OGP via the alternative translation product H4-(84-102), but not whole histone H4.

Histogranin-like antinociceptive and anti-inflammatory derivatives of o-phenylenediamine and benzimidazole.

Histogranin (HN)-like nonpeptides were designed and synthesized using benzimidazole (compound 1) and o-phenylenediamine (compounds 2-7) as scaffolds for the attachment of phenolic hydroxyl and basic guanidino pharmacophoric elements present in HN. The benzimidazole derivative N-5-guanidinopentanamide-(2R)-yl-2-(p-hydroxybenzyl)-5-carboxybenzimidazole (1) and the o-phenylenediamine derivative N-5-guanidinopentanamide-(2S)-yl-2-N-(p-hydroxyphenylacetyl) phenylenediamine (2) were more potent analgesics than HN in both the mouse writhing (5.5 and 3.5 as potent as HN, respectively) and tail-flick (11.8 and 8.0 as potent as HN, respectively) pain assays. Improvements in the potencies and times of action of compound 2 in the mouse writhing test were obtained by attaching carboxyl (6)or p-Cl-benzoyl (7) groups at position 4 of the (2R) o-phenylenediamine derivative (5). In rats, compounds 2 (80 nmol i.t.), 6 (36 nmol i.t.), and 7 (18 nmol i.t.) were effective in blocking both persistent inflammatory pain in the formalin test and hyperalgesia in the complete Freund adjuvant assay. Compounds 2, 6, and 7, but not compound 1 at 10 nmol (i.c.v.) also mimicked the HN (60 nmol i.c.v.) blockade of N-methyl-D-aspartate (NMDA)-induced convulsions in mice. Finally, in primary cultures of rat alveolar macrophages, HN and compounds 1, 2, 6, and 7 (10(-8) M) significantly blocked lipopolysaccharide-induced cyclooxygenase-2 induction and prostaglandin E(2) secretion. These studies indicate that both derivatives of benzimidazole and o-phenylenediamine mimic the in vivo antinociceptive and in vitro anti-inflammatory effects of HN, but the HN protection of mice against NMDA-induced convulsions is mimicked only by the o-phenylenediamine derivatives.

Bioactive peptidic analogues and cyclostereoisomers of the minimal antinociceptive histogranin fragment-(7-10).

Novel analogues of the minimal antinociceptive histogranin (HN) fragment Gly(7)-Gln-Gly-Arg(10), in which amino acids in positions 8, 9, and 10 were replaced by lipophilic amino acids and corresponding d-amino acid residues in combination with N- to C-terminal cyclization, were synthesized and tested in various animal models of pain. All synthetic compounds were potent and efficacious analgesics in the mouse writhing test. Cyclic [-Gly-Ala-Tyr-d-Arg-] (9) and cyclic [-Gly p-Cl-Phe-Tyr-d-Arg-] (10) were the most potent analgesics, being 17 and 135 times as potent as HN, respectively (AD(50) of 1.37 and 0.17 nmol/mouse icv, as compared with 23 nmol/mouse for HN). The times of action of compounds 9 and 10 were also much improved with half-maximal effects still being observed 60 min and >90 min after their administration, respectively, as compared with 8.1 min for the parent peptide HN-(7-10) and 22.1 min for HN. At analgesic doses, compounds 9 and 10 were devoid of motor effect as assessed by the mouse rotarod assay. As already observed with HN, compounds 9 (10 nmol/rat; i.t.) and 10 (0.5 nmol/rat; i.t.) were effective in blocking persistent inflammatory pain in the formalin test and hyperalgesia induced by intraplantar administration of complete Freund adjuvant. In addition, the analgesic effects evoked by compounds 9 (10 nmol/mouse; icv) and 10 (1 micromol/kg; i.v.) in the mouse writhing test and compound 9 (10 nmol/mouse; icv) in the mouse tail flick assay were similarly antagonized by the dopamine D(2) receptor antagonist raclopride (1 nmol/mouse; icv) but not the opiate antagonist naloxone (1 nmol/mouse; i.c.v). Finally, the various cyclic compounds competed with the binding of [(3)H]raclopride in rat brain membrane preparations. Their ability to compete with the binding of the D(2) ligand correlated well with their potency in alleviating pain in the mouse writhing test (r = 0.95). These results indicate that the analgesic activity of the minimal active core in HN can be improved by changes that favor its interaction with the dopamine D(2) receptor.