Peptides (v.22, #6)

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi, a protozoan parasite that is the causative antigen of Chagas’ disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi-infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10–50 μM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.

Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1 by Patrizia M. Germano; James Stalter; Sang V. Le; Mark Wu; Dean J. Yamaguchi; David Scott; Joseph R. Pisegna (861-866).
Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.
Keywords: PACAP; Confocal microscopy; Fluo-PACAP; PAC1; NIH; 3T3 cells; Pharmacology;

The neuromodulatory effects of VIP/PACAP on PC-12 cells are associated with their N-terminal structures by Satomi Onoue; Yoshihiro Waki; Yumiko Nagano; Seiji Satoh; Kazuhisa Kashimoto (867-872).
ONOUE, S., WAKI, Y., NAGANO, Y., SATOH, S., KASHIMOTO, K. Neuromodulatory Effects of VIP/PACAP on PC-12 Cells Are Associated with Their N-terminal Structures. PEPTIDES xx(xx) 000–000, 200x.– The current study explored whether the differences in biological activities in PC-12 cells between vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are attributable to the sequence difference in their N-terminal portions and are correlated with the solution structures of the peptides. In the neurite outgrowth assay, N-terminal modification of VIP to PACAP-like sequences altered its effect, the activity was confirmed even at a low concentration (10−10 M). On the contrary, N-terminal modification of PACAP 27 to VIP-like sequences reduced its activity. These relationships were also confirmed for the inhibitory effects of the peptide analogues on PC-12 cells growth at 10−7 M. The present results combined with our previously reported data, including binding assay, support that the N-termini of VIP/PACAP plays an important role in their activities.
Keywords: VIP; PACAP; PC-12 cells; Neurite outgrowth; Neuropeptides; Molecular dynamics; Structure-activity relationships;

The levels of the pituitary adenylate cyclase activating polypeptide (PACAP) were measured in the central nervous system and in peripheral organs of the anoxia-tolerant freshwater turtle, Pseudemys scripta elegans by radioimmunoassay. The concentration of PACAP38 was strikingly high in the central nervous system and lower but considerable immunoreactivity was detected in the peripheral organs. Levels of PACAP38 in the turtle brain exceed those measured in rat and human brain areas by 10–100-fold. Based on these exceptionally high levels of PACAP and the known neuroprotective role of the peptide, it can be suggested that PACAP38 plays a role in the extraordinary resistance of the turtle brain from anoxia-induced neuronal damage.
Keywords: PACAP; Turtle; Anoxia-tolerance; Neuroprotection;

Antiproliferative actions of growth hormone-releasing hormone antagonists on MiaPaCa-2 human pancreatic cancer cells involve cAMP independent pathways by Zoltan Rekasi; Jozsef L. Varga; Andrew V. Schally; Artur Plonowski; Gabor Halmos; Balazs Csernus; Patricia Armatis; Kate Groot (879-886).
We evaluated the effects of GHRH antagonists on the proliferation of MiaPaCa-2 human pancreatic cancer cells and cAMP signaling in vitro. GHRH antagonists inhibited the proliferation of MiaPaCa-2 cells in vitro in a dose-dependent way and caused a significant elevation in cAMP production. In a superfusion system, short-term exposure of the cells to GHRH antagonists evoked an acute, dose-dependent release of cAMP into the medium. Native GHRH, which stimulates cAMP efflux from pituitary at nanomolar doses, did not influence cAMP release from cultured or superfused MiaPaCa-2 cells even at 10–30 μM. VIP, PACAP, secretin and glucagon also did not influence cell proliferation or cAMP production. Adenylate cyclase activator forskolin (FSK) caused a greater cAMP response, but a smaller antiproliferative effect than GHRH antagonists. Combined treatment with FSK and GHRH antagonist JV-1-38 potentiated the cAMP-inducing effect of FSK, but did not produce a greater inhibition of cell proliferation than JV-1-38 alone. A selective accumulation of radiolabeled GHRH antagonist [125I]JV-1-42 in vivo in MiaPaCa-2 carcinoma xenografted into nude mice was also observed. In conclusion, second messengers other than cAMP participate in the signal transduction pathways of GHRH analogs mediated by tumoral GHRH receptors.
Keywords: GHRH antagonists; Tumoral GHRH receptors; Signal transduction; cAMP; Anticancer drugs;

Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y4 or Y5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y4 receptors are also selectively blocked by human or rat pancreatic polypeptide (PP) and the blocking peptides are not dissociated by high concentrations of alkali chlorides (which restore most of the binding of subtype-selective agonists to Y1 and Y2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y1-selective agonists (ic 50 300–400 pM). The cloned Y4 receptors from three species are much less sensitive to NPY or PYY. The sensitivity of both the PP/NPY sites and the Y4 sites to Y2-selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y1 antagonist ((Cys31,NVal34NPY(27-36))2, which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na+ transport, and by RFamide NRNFLRF.NH2, but not by Ca2+ channel blockers, or by inhibitors of K+ transport. Protection of both PP/NPY and Y4 sites against blockade by human or rat pancreatic polypeptide is also afforded by short N-terminally truncated NPY-related peptides. The above results are consistent with a stringent and selective activity regulation for rabbit PP/NPY receptor(s) that may serve to differentiate agonists and constrain signaling, and could involve transporter-like interactants.
Keywords: Cation sensitivity; Amiloride derivative; RFamide;

Diazoxide effects on hypothalamic and extra-hypothalamic NPY content in Zucker rats by I.E Hensley; J.E Lawler; R Alemzadeh; S.J Holshouser (899-908).
To determine if the anorectic effects of the insulin antagonist diazoxide (DZ) are mediated by reduced central neuropeptide Y (NPY), female Zucker rats, given DZ (150 mg/kg/day) or placebo for about four weeks, were sacrificed following overnight fasting or free feeding. Several hypothalamic and extra-hypothalamic nuclei were extracted for NPY content. DZ reduced weight gain in obese rats and lowered glucose of lean and obese rats without affecting insulin. Contrary to the hypothesis, DZ increased NPY in hypothalamic nuclei of free fed lean and obese rats. DZ elevated hypothalamic NPY levels in fasted obese rats and had more diverse effects in extra-hypothalamic nuclei of lean rats.
Keywords: Neuropeptide Y; Insulin; Diazoxide; Hypothalamus; Zucker rat; Radioimmunoassay; Micropunch procedure; Obesity;

We recently reported the direct inhibitory effect of adrenomedullin on caecal circular smooth muscle cells via cAMP system. This study was designed to determine whether the structurally related peptides to adrenomedullin (i.e.; calcitonin gene-related peptide (CGRP), calcitonin, and amylin) can inhibit the cholecystokinin octapeptide (CCK-8)-induced contractile response by exerting a direct action on guinea-pig caecal circular smooth muscle cells, and to compare the inhibitory potency of these peptides. In addition, to elucidate each intracellular mechanisms, the effects of an inhibitor of cAMP-dependent protein kinase, inhibitors of particulate or soluble guanylate cyclase on the each peptide-induced relaxation were investigated. Adrenomedullin, CGRP, calcitonin, and amylin inhibited the contractile response produced by CCK-8 in a dose-dependent manner, with IC50 values of 0.14 nM, 0.37 nM, 5.4 nM, and 160 nM, respectively. An inhibitor of cAMP-dependent protein kinase significantly inhibited the relaxation produced by all of these peptides. On the contrary, inhibitors of particulate or soluble guanylate cyclase did not have any significant effect on the relaxation produced by these peptides. In this study, we demonstrated the direct inhibitory effects of the structurally related peptides to adrenomedullin (i.e.; CGRP, calcitonin, and amylin) on the isolated caecal circular smooth muscle cells via cAMP system. The order of potency was as follows; adrenomedullin ≒ CGRP > calcitonin > amylin.
Keywords: Adrenomedullin; Amylin; Calcitonin; CGRP; Guinea-pig; Isolated circular smooth muscle cells;

The involvement of kinins, calcitonin gene-related peptide (CGRP), and tachykinins during mesenteric post-ischemic reperfusion was studied in anesthetized rats by using antagonists for bradykinin (BK) B1, BK B2, CGRP1, or tachykinin NK1 receptor, or by capsaicin-induced desensitization. B1, B2, or CGRP1 receptor antagonists or desensitization attenuated the transient hypotension and plasma protein and leukocyte infiltration of intestinal wall observed during post-ischemic reperfusion. These effects were abolished by the combination of B2 and CGRP1 blockade as well as by B2 antagonism in capsaicinized rats, while NK1 blockade was ineffective. Our results suggest that kinins and CGRP contribute to systemic vasodilatation and microvascular leakage during mesenteric reperfusion. Pharmacological blockade of these systems could help preventing hypotension and intestinal injury consequent to reperfusion.
Keywords: Intestinal reperfusion; Kinins; Calcitonin gene-related peptide; Hypotension; Plasma extravasation;

Stimulation of endogenous nitric oxide production is involved in the inhibitory effect of adrenomedullin on aldosterone secretion in the rat by Piera Rebuffat; Ludwik K Malendowicz; Gastone G Nussdorfer; Giuseppina Mazzocchi (923-926).
Adrenomedullin (AM) (10−8 M) partially suppressed aldosterone response of dispersed rat zona glomerulosa (ZG) cells to 10 mM K+, and the nitric oxide (NO) synthase inhibitors L-NAME (10−3 M) and 1400W (10−4 M) effectively counteracted this effect of AM. The NO donor L-Arginine (L-Arg) (10−5 M) decreased both basal and K+-stimulated aldosterone secretion. The guanylate-cyclase inhibitor Ly-83583, at a concentration (10−4 M) abolishing either the guanylate-cyclase activator guanylin- or L-Arg-induced cGMP release from dispersed ZG cells, did not affect the aldosterone antisecretagogue action of AM and L-Arg. AM (10−8 M) evoked a moderate increase in cGMP release by dispersed ZG cells, and the effect was blocked by both 10−4 M Ly-83583 and 10−3 M L-NAME. Collectively, these findings allow us (1) to confirm that NO inhibits aldosterone secretion through a cGMP-independent mechanism; and (2) to suggest that stimulation of endogenous NO synthesis plays a role in the mechanisms underlying the inhibitory effect of AM on K+-stimulated aldosterone secretion from rat ZG cells.
Keywords: Adrenomedullin; Nitric oxide; cGMP; Aldosterone; Rat (Sprague-Dawley);

Gender differences in the attenuation of salt-induced hypertension by angiotensin (1-7) by Danita Eatman; Min Wang; Robin R. Socci; Myrtle Thierry-Palmer; Nerimiah Emmett; Mohamed A. Bayorh (927-933).
Chronic infusion of angiotensin (1-7) [Ang-(1-7)] lowers blood pressure in spontaneously hypertensive rats (SHR). To assess the role of Ang-(1-7) in salt-induced hypertension, Ang-(1-7) (24 μg/kg/hr) or saline was administered chronically via osmotic minipump into the jugular vein of 5–6 wk-old male (M) and female (F) Dahl salt-sensitive rats placed on a high-salt (8% NaCl) diet for 2 weeks. Blood pressure (BP) and heart rate were measured prior to the start of the diet and weekly thereafter. Ang-(1-7) significantly attenuated the BP increase after 1 wk on the diet in both M and F rats, but after 2 weeks only in F rats. Enhanced release of prostacyclin, (6-keto PGF), following Ang-(1-7) treatment was observed in both M and F rats. In addition, significant increases in aortic blood flow and plasma levels of nitric oxide were observed in the F rats following Ang-(1-7) treatment. These findings demonstrate that the reduction in BP is due to both prostacyclin and NO and that there is a gender difference in the attenuation of salt-induced hypertension by Ang-(1-7).
Keywords: Dahl rats; Angiotensin-(1-7); Blood pressure; Gender; Prostaglandins; Nitric oxide; Blood flow;

A role for the angiotensin IV/AT4 system in mediating natriuresis in the rat by T.A Hamilton; R.K Handa; J.W Harding; J.W Wright (935-944).
Angiotensin II (AngII) or Angiotensin IV (AngIV) was infused into the renal artery of anesthetized rats while renal cortical blood flow was measured via laser Doppler flowmetry. The infusion of AngII produced a significant elevation in mean arterial pressure (MAP) with an accompanying decrease in cortical blood flow, glomerular filtration rate (GFR), urine volume, and urine sodium excretion. The infusion of AngIV induced significant increases in renal cortical blood flow and urine sodium excretion, without altering MAP, GFR, and urine volume. Pretreatment infusion with a specific AT1 receptor antagonist, DuP 753, blocked or attenuated the subsequent AngII effects, while pretreatment infusion with the specific AT4 receptor antagonist, Divalinal-AngIV, blocked the AngIV effects. These results support distinct and opposite roles for AngII and AngIV, i.e. AngII acts as an anti-natriuretic agent, while AngIV acts as a natriuretic agent.
Keywords: Angiotensin II; Angiotensin IV; AT1 receptor; AT4 receptor; Kidney cortical blood flow; Glomerular filtration rate; Urine sodium excretion; DuP 753; Divalinal-AngIV;

Angiotensinase activity in mice fed an olive oil-supplemented diet by Marı́a Jesús Ramı́rez-Expósito; José Manuel Martı́nez-Martos; Isabel Prieto; Francisco Alba; Manuel Ramı́rez (945-952).
We evaluated the influence of a diet supplemented with olive oil (20% by weight) (OO) on the activity of glutamyl aminopeptidase (GluAP) and aspartyl aminopeptidase (AspAP), which are involved in angiotensin metabolism. Serum concentrations of total cholesterol and fatty acids were also measured. Animals fed on the OO diet gained significantly more weight than did controls from the second week until the end of the feeding period. Serum total cholesterol concentration was significantly higher in the OO group than in control mice. Total monounsaturated fatty acids increased in OO-fed animals, but total saturated fatty acids decreased. No differences between the two groups were observed for total polyunsaturated fatty acids. Serum from animals fed on the OO diet contained significantly lower proportions of myristic, pentadecanoic, palmitic, palmitoleic, vaccenic, α-linolenic, γ-linolenic, and 11,14-eicosadienoic acids than did serum from control animals. In contrast, the OO group had higher levels of oleic, stearic, and gondoic acids. GluAP activity decreased significantly in the serum of OO-fed animals. In these animals soluble AspAP activity was significantly higher in the testis, and significantly lower in the lung and adrenal, in comparison to controls. Membrane-bound AspAP activity was higher in the testis and atrium, and lower in lung, in the OO group. Soluble GluAP activity was significantly lower in the testis of OO-fed animals. Membrane-bound GluAP activity did not differ between the two groups in any of the tissues analyzed. Serum AspAP and GluAP activities correlated negatively with palmitoleic and vaccenic acid respectively in the OO group. However, no significant correlations were found in the control group. These results may reflect functional changes in the renin-angiotensin system in the serum, adrenal, testis, lung and atrium after feeding with a diet enriched in olive oil.
Keywords: Aminopeptidases; Angiotensinases; Olive oil; Cholesterol; Fatty acids;

Effects of the vasopeptidase inhibitor omapatrilat on cardiac endogenous kinins in rats with acute myocardial infarction by Charles Blais; Nathalie Lapointe; Jean-Lucien Rouleau; Robert Clément; Nicole Gervais; David Geadah; Albert Adam (953-962).
The purposes of this study were to evaluate and to compare the effects of simultaneous angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) inhibition by the vasopeptidase inhibitor omapatrilat (1 mg · kg−1 · day−1) with those of the selective ACE inhibitor enalapril (1 mg · kg−1 · day−1) on survival, cardiac hemodynamics, and bradykinin (BK) and des-Arg9-BK levels in cardiac tissues 24 h after myocardial infarction (MI) in rats. The effect of the co-administration of both B1 and B2 kinin receptor antagonists (2.5 mg · kg−1 · day−1 each) with metallopeptidase inhibitors was also evaluated. The pharmacological treatments were infused subcutaneously using micro-osmotic pumps for 5 days starting 4 days before the ligation of the left coronary artery. Immunoreactive kinins were quantified by highly sensitive and specific competitive enzyme immunoassays. The post-MI mortality of untreated rats with a large MI was high; 74% of rats dying prior to the hemodynamic study. Mortality in the other MI groups was not significantly different from that of the untreated MI rats. Cardiac BK levels were not significantly different in the MI vehicle–treated group compared with the sham–operated rats. Both omapatrilat and enalapril treatments of MI rats significantly increased cardiac BK concentrations compared with the sham–operated group (P < 0.05). However, cardiac BK levels were significantly increased only in the MI omapatrilat–treated rats compared with the MI vehicle–treated group (P < 0.01). Cardiac des-Arg9-BK concentrations were not significantly modified by MI, and MI with omapatrilat or enalapril treatment compared with the sham-operated group. The co-administration of both kinin receptor antagonists with MI omapatrilat– and enalapril–treated rats had no significant effect on cardiac BK and des-Arg9-BK levels. Thus, the significant increase of cardiac BK concentrations by omapatrilat could be related to a biochemical or a cardiac hemodynamic parameter on early (24 h) post-MI state.
Keywords: Angiotensin-converting enzyme; Bradykinin; des-Arg9-bradykinin; Heart; Metallopeptidase inhibition; Myocardial infarction; Neutral endopeptidase 24.11;

Muscle layer- and region-dependent distributions of oxytocin receptors in the porcine myometrium by Takio Kitazawa; Taku Kajiwara; Akira Kiuchi; Hirofumi Hatakeyama; Tetsuro Taneike (963-974).
The aim of the present study was to clarify smooth muscle- and region-dependent distributions of the oxytocin receptor that mediates oxytocin-induced contraction in the nonpregnant porcine myometrium by means of mechanical and radioligand ([3H]-oxytocin) binding studies. In Krebs solution, oxytocin (0.1–300 nM) caused concentration-dependent contractions of the cornual myometrium, and the longitudinal muscle was more sensitive than the circular muscle. [Arg8]-vasopressin and [deamino-Cys1, D-Arg8]-vasopressin also contracted the myometrium, and the order of the potency was oxytocin > [Arg8]-vasopressin > [deamino-Cys1, D-Arg8]-vasopressin. Treatment with a high concentration of oxytocin selectively inhibited the contraction of oxytocin and [Arg8]-vasopressin without affecting the responses of acetylcholine and high-K+. Selective cross inhibition was also observed in the presence of a high concentration of [Arg8]-vasopressin. The oxytocin-induced contraction was resistant to tetrodotoxin and atropine, but was reduced by verapamil or by the removal of external Ca2+, indicating that oxytocin has a direct action on smooth muscle cells and that extracellular Ca2+ plays an important role for the contraction. In Kumagai solution, oxytocin caused contraction of the cornual longitudinal muscle (−logEC50 = 8.5) but not the circular muscle. Longitudinal muscles of other regions (corpus and cervix) were also responsive to oxytocin, but the −logEC50 value differed from region to region (cornua > corpus = cervix). On the other hand, oxytocin failed to cause contraction of the corpus and cervical circular muscles. 3H-Oxytocin bound to crude membrane preparations of the myometrium in a concentration-dependent (0.084–2.7 nM) saturable manner. Scatchard analysis of equilibrium binding data revealed the presence of a single class of binding site with an apparent dissociation constant (Kd, 1.1–1.5 nM), but receptor density (Bmax) differed in the two muscle layer types (longitudinal muscle: circular muscle = 5:1) and tended to decrease from the cornua to the cervix. In conclusion, the receptor specific for oxytocin is present in the porcine myometrium and mediates the contractile responses of both oxytocin and [Arg8]-vasopressin. The distribution of the oxytocin receptors differs according to the type of muscle layer (longitudinal muscle > circular muscle) and the region of the uterus.
Keywords: Oxytocin; [Arg8]-vasopressin; Oxytocin receptor; Porcine myometrium; Contraction;

Chromatin-derived acidic peptides modulate catecholamine release in the hypothalamus☆ by Luigi Brunetti; Giustino Orlando; Barbara Michelotto; Lucia Recinella; Michele Vacca (975-978).
We have studied the neuromodulatory effects of three synthetic peptides, structurally related to chromatin-derived acidic peptides (ACPs): ACP-1 (Asp-Asp-Ser-Asp-Glu-Glu-Asn), corresponding to the C-terminal fragment of the largest subunit of eukaryotic RNA polymerase II; a more lipophilic derivative, ACP-2 (Ala-Ile-Ser-Pro-Asp-Asp-Ser-Asp-Glu-Glu-Asn); and its phosphorylated form ACP-3 (Ala-Ile-Ser-Pro-Asp-Asp-Ser(P)-Asp-Glu-Glu-Asn). Rat hypothalamic synaptosomes, loaded with [3H]norepinephrine or [3H]dopamine, were perfused with the above peptides, both basally and during a depolarizing stimulus. We have found: ACP-1 inhibited both dopamine and norepinephrine release; ACP-2 inhibited dopamine release, without affecting norepinephrine release; ACP-3 was almost ineffective, except for a weak dopamine inhibiting effect only at a higher concentration.
Keywords: Hypothalamus; Catecholamine; Dopamine; Norepinephrine; Chromatin-derived acidic peptides;

N-Acetylcarnosine, a natural histidine-containing dipeptide, as a potent ophthalmic drug in treatment of human cataracts by Mark A. Babizhayev; Anatoly I. Deyev; Valentina N. Yermakova; Yuri A. Semiletov; Nina G. Davydova; Natalya I. Kurysheva; Alexander V. Zhukotskii; Ita M. Goldman (979-994).
A study was designed to document and quantify the changes in lens clarity over 6 and 24 months in 2 groups of 49 volunteers (76 eyes) with an average age of 65.3 ± 7.0 enrolled at the time of diagnosis of senile cataracts of minimal to advanced opacification.The patients received N-acetylcarnosine, 1% sol (NAC) (26 patients, 41 eyes = Group II), placebo composition (13 patients, 21 eyes) topically (two drops, twice daily) to the conjunctival sac, or were untreated (10 patients, 14 eyes); the placebo and untreated groups were combined into the control (reference) Group I. Patients were evaluated upon entry, at 2-month (Trial 1) and 6-month (Trial 2)-intervals for best corrected visual acuity (b/c VA), by ophthalmoscopy and the original techniques of glare test (for Trial 1), stereocinematographic slit-image and retro-illumination photography with subsequent scanning of the lens. The computerized interactive digital analysis of obtained images displayed the light scattering/absorbing centers of the lens into 2-D and 3-D scales.The intra-reader reproducibility of measuring techniques for cataractous changes was good, with the overall average of correlation coefficients for the image analytical data 0.830 and the glare test readings 0.998. Compared with the baseline examination, over 6 months 41.5% of the eyes treated with NAC presented a significant improvement of the gross transmissivity degree of lenses computed from the images, 90.0% of the eyes showed a gradual improvement in b/c VA to 7–100% and 88.9% of the eyes ranged a 27–100% improvement in glare sensitivity. Topographic studies demonstrated less density and corresponding areas of opacification in posterior subcapsular and cortical morphological regions of the lens consistent with VA up to 0.3. The total study period over 24 months revealed that the beneficial effect of NAC is sustainable. No cases resulted in a worsening of VA and image analytical readings of lenses in the NAC-treated group of patients. In most of the patients drug tolerance was good. Group I of patients demonstrated the variability in the densitometric readings of the lens cloudings, negative advance in glare sensitivity over 6 months and gradual deterioration of VA and gross transmissivity of lenses over 24 months compared with the baseline and 6-month follow-up examinations. Statistical analysis revealed the significant differences over 6 and 24 months in cumulative positive changes of overall characteristics of cataracts in the NAC-treated Group II from the control Group I.The N-acetylated form of natural dipeptide L-carnosine appears to be suitable and physiologically acceptable for nonsurgical treatment for senile cataracts.
Keywords: Antioxidant; Natural product dipeptide; N-acetylcarnosine; L-carnosine; Ophthalmic drug; Lens; Cataract; Ocular administration;

Neuronal messengers in the human cerebral circulation by Sérgio Gulbenkian; Rolf Uddman; Lars Edvinsson (995-1007).
In recent years our knowledge of the nervous control of the cerebral circulation has increased. The use of denervations and retrograde tracing in combination with immunohistochemical techniques has demonstrated that cerebral vessels are supplied with sympathetic, parasympathetic, and sensory nerve fibers and possibly central pathways containing a multiplicity of new transmitter substances in addition to the classical transmitters. The majority of these transmitters are neuropeptides. More recently it has been suggested that a gaseous transmitter, nitric oxide (NO) also could participate in the neuronal regulation of cerebral blood flow. Although little is known about the physiological actions and inter-relationships among all these putative neurotransmitters, their presence within cerebrovascular nerve fibers will make it necessary to revise our view on the mechanisms of cerebrovascular neurotransmission.