Distinct tumor-infiltrating lymphocyte landscapes are associated with clinical outcomes in localized non-small-cell lung cancer.

BACKGROUND: Despite the importance of tumor-infiltrating T lymphocytes (TILs) in cancer biology, the relationship between TIL phenotypes and their prognostic relevance for localized non-small-cell lung cancer (NSCLC) has not been well established. PATIENTS AND METHODS: Fresh tumor and normal adjacent tissue was prospectively collected from 150 patients with localized NSCLC. Tissue was comprehensively characterized by high-dimensional flow cytometry of TILs integrated with immunogenomic data from multiplex immunofluorescence, T-cell receptor sequencing, exome sequencing, RNA sequencing, targeted proteomics, and clinicopathologic features. RESULTS: While neither the magnitude of TIL infiltration nor specific TIL subsets were significantly prognostic alone, the integration of high-dimensional flow cytometry data identified two major immunotypes (IM1 and IM2) that were predictive of recurrence-free survival independent of clinical characteristics. IM2 was associated with poor prognosis and characterized by the presence of proliferating TILs expressing cluster of differentiation 103, programmed cell death protein 1, T-cell immunoglobulin and mucin-domain containing protein 3, and inducible T-cell costimulator. Conversely, IM1 was associated with good prognosis and differentiated by an abundance of CD8+ T cells expressing cytolytic enzymes, CD4+ T cells lacking the expression of inhibitory receptors, and increased levels of B-cell infiltrates and tertiary lymphoid structures. While increased B-cell infiltration was associated with good prognosis, the best prognosis was observed in patients with tumors exhibiting high levels of both B cells and T cells. These findings were validated in patient tumors from The Cancer Genome Atlas. CONCLUSIONS: Our study suggests that although the number of infiltrating T cells is not associated with patient survival, the nature of the infiltrating T cells, resolved in distinct TIL immunotypes, is prognostically relevant in NSCLC and may inform therapeutic approaches to clinical care.

Effects of slow- v. fast-digested protein supplementation combined with mixed power training on muscle function and functional capacities in older men.

Ageing leads to a progressive loss of muscle function (MF) and quality (MQ: muscle strength (MS)/lean muscle mass (LM)). Power training and protein (PROT) supplementation have been proposed as efficient interventions to improve MF and MQ. Discrepancies between results appear to be mainly related to the type and/or dose of proteins used. The present study aimed at determining whether or not mixed power training (MPT) combined with fast-digested PROT (F-PROT) leads to greater improvements in MF and MQ in elderly men than MPT combined with slow-digested PROT (S-PROT) or MPT alone. Sixty elderly men (age 69 (sd 7) years; BMI 18-30 kg/m2) were randomised into three groups: (1) placebo + MPT (PLA; n 19); (2) F-PROT + MPT (n 21) and (3) S-PROT + MPT (n 20) completed the intervention. LM, handgrip and knee extensor MS and MQ, functional capacity, serum metabolic markers, skeletal muscle characteristics, dietary intake and total energy expenditure were measured. The interventions consisted in 12 weeks of MPT (3 times/week; 1 h/session) combined with a supplement (30 g:10 g per meal) of F-PROT (whey) or S-PROT (casein) or a placebo. No difference was observed among groups for age, BMI, number of steps and dietary intake pre- and post-intervention. All groups improved significantly their LM, lower limb MS/MQ, functional capacity, muscle characteristics and serum parameters following the MPT. Importantly, no difference between groups was observed following the MPT. Altogether, adding 30 g PROT/d to MPT, regardless of the type, does not provide additional benefits to MPT alone in older men ingesting an adequate (i.e. above RDA) amount of protein per d.

Muscle adaptation in response to a high-intensity interval training in obese older adults: effect of daily protein intake distribution.

BACKGROUND: Aging is associated with declines in muscle mass, strength and quality, leading to physical impairments. An even protein distribution in daily meals has recently been proposed along with adequate total protein intake as important modulators of muscle mass. In addition, due to its short duration, high-intensity interval training (HIIT) has been highlighted as a promising intervention to prevent physical deterioration. However, the interaction between daily protein intake distribution and HIIT intervention in elderlies remain unknown. OBJECTIVE: To investigate muscle adaptation following HIIT in older adults according to daily protein intake distribution. METHODS: Thirty sedentary obese subjects who completed a 12-week elliptical HIIT program were matched [criteria: age (± 2 years), sex, BMI (± 2 kg/m2)] and divided a posteriori into 2 groups according to the amount of protein ingested at each meal: < 20 g in at least one meal (P20-, n = 15, 66.8 ± 3.7 years) and ≥ 20 g in each meal (P20+, n = 15, 68.1 ± 4.1 years). Body composition, functional capacity, muscle strength, muscle power, physical activity level, and nutritional intakes were measured pre- and post-intervention. A two way repeated ANOVA was used to determine the effect of the intervention (HIIT) and protein distribution (P20- vs P20+, p < 0.05). RESULTS: No difference was observed at baseline between groups. Following the HIIT intervention, we observed a significant decrease in waist and hip circumferences and improvements in functional capacities in both P20- and P20 + group (p < 0.05). However, no protein distribution effect was observed. CONCLUSION: A 12-week HIIT program is achievable and efficient to improve functional capacities as well as body composition in obese older adults. However, consuming at least 20 g of proteins in every meal does not further enhance muscle performance in response to a 12-week HIIT intervention.

Cerebrovascular hemodynamic correlates of aging in the Lou/c rat: a model of healthy aging.

The LOU/c rat is an inbred strain considered a model of healthy aging. It exhibits a longer free disease lifespan and a low adiposity throughout life. While this animal model has been shown to maintain eating behavior and neuroendocrine, metabolic and cognitive functions with age, no study has yet investigated vascular correlates in this model of healthy aging. In the present work, multispectral optical imaging was used to investigate the hemodynamic response in the somatosensory cortex of LOU/c rats following forepaw stimulation in three age groups, 4, 24 and 40months. Results indicate reduced hemodynamic responses in the contralateral somatosensory cortex between young (4months) and older groups following stimulation. This decrease was associated with an increase in the spatial extent of activation. The ipsilateral response did not change with aging leading to decreased laterality. Estimations of the relative change in the local cerebral metabolic rate of oxygen during stimulation based on multimodal data showed no significant change with age. The exponent describing the relation between blood volume and blood flow changes, Grubb's parameter, did display a significant change with age which may suggest vessel compliance modifications. This work finds its relevance in recent findings underlying the importance of vascular changes with aging and its impact on neurodegenerative disease.

Biophysical model estimation of neurovascular parameters in a rat model of healthy aging.

Neuronal, vascular and metabolic factors result in a deterioration of the cerebral hemodynamic response with age. The interpretation of neuroimaging studies in the context of aging is rendered difficult due to the challenge in untangling the composite effect of these modifications. In this work we integrate multimodal optical imaging in biophysical models to investigate vascular and metabolic changes occurring in aging. Multispectral intrinsic optical imaging of an animal model of healthy aging, the LOU/c rat, is used in combination with somatosensory stimulation to study the modifications of the hemodynamic response with increasing age. Results are fitted with three macroscopic biophysical models to extract parameters, providing a phenomenological description of vascular and metabolic changes. Our results show that 1) biophysical parameters are estimable from multimodal data and 2) parameter estimates in this population change with aging.

Differences in muscle adaptation to a 12-week mixed power training in elderly men, depending on usual protein intake.

INTRODUCTION: Normal aging is often associated with a decline of muscle mass (MM), strength (MS) and quality (MQ: MS/MM), leading to functional incapacities. This aging-related deterioration of muscles may involve a decreased protein intake. Mixed power training has been recently shown to induce positive effects on MM, MS and MQ. However, to our knowledge, no study has examined if muscle adaptations following mixed power training could be influenced by the daily amount of protein ingested in elderly men. METHODS: Twenty-one men completed the intervention and were divided into 2 groups based on their usual protein intake: PROT 1.1- (<1.1 g·kg-1·d-1 [n = 10; 73 ±â€¯3 years]) and PROT 1.2+ (>1.2 g·kg-1·d-1 [n = 11; 73 ±â€¯3 years]). Body composition (DXA: lean and fat masses), MS (1-maximal repetition on leg-press and handgrip strength), MQ (MS/body mass and MS/lower limb lean mass), functional capacities (Short Physical Performance Battery/Senior Fitness Test), dietary intake (3-day food record) and energy expenditure (accelerometer; 7 days) were measured. Mixed power training intervention consisted in power and functional exercises (12 weeks; 3 times/week; 1 h/session). RESULTS: Lower limb MS increase in the PROT 1.2+ group was greater from that of the PROT 1.1- group when normalized to lower limbs lean mass (p = 0.036). In addition, a trend for greater gain in lower limb MS normalized to body mass (p = 0.053) was observed in the PROT 1.2+. CONCLUSION: To optimize mixed power training effects on muscle function, healthy older men should ingest daily at least 1.2 g·kg-1·d-1 of protein. These beneficial effects of a higher usual protein intake were observed especially for MQ, which is one of the best predictors of functional capacities in older adults.

Intakes of folate, vitamin B6 and B12 and risk of depression in community-dwelling older adults: the Quebec Longitudinal Study on Nutrition and Aging.

BACKGROUND/OBJECTIVES: Depression can decrease quality of life and affect health outcomes in older population. We investigated whether different intake levels of folate, vitamin B6 and B12 were associated with a 3-year depression incidence among generally healthy, community-dwelling older men and women. SUBJECTS/METHODS: Participants in the Québec Longitudinal Study on Nutrition and Aging (NuAge), free of depression (that is, 30-item Geriatric Depression Scale (GDS) <11) at baseline (N=1368; 74 ± 4 years old; 50.5% women), were screened annually for incident depression (GDS ⩾ 11) or antidepressant medication. Tertiles of intakes (food only and food+supplements) were obtained from the mean of three non-consecutive 24-h recalls at baseline. Sex-stratified multiple logistic regression models were adjusted for age, physical activity, physical functioning, stressful life events and total energy intake. RESULTS: Over 3 years, 170 participants were identified as depressed. Women in the highest tertile of B6 intake from food were 43% less likely to become depressed when adjusting for demographic and health factors (multivariate odds ratio (OR) 0.57, 95% confidence interval (CI) 0.39-0.96), but adjustment for energy intake attenuated the effect. Men in the highest tertile of dietary B12 intake had decreased risk of depression (energy-adjusted multivariate OR 0.42, 95% CI 0.20-0.90). No other association was observed. CONCLUSIONS: This study provides some evidence of decreased depression risk among women with higher intakes of vitamin B6 from food, which was dependent on total energy intake, and among men with higher intakes of B12 from food, independently of energy intake.

Muscle Mass Index and Animal Source of Dietary Protein Are Positively Associated with Insulin Resistance in Participants of the NuAge Study.

OBJECTIVES: Contribute evidence towards the complex interrelationships of body composition, insulin sensitivity and protein intake independently from adiposity in an older population. DESIGN: This is a cross-sectional analysis of an existing dataset in which a literature-supported model linking together the variables of interest is tested using path analysis. SETTING: The loss of muscle mass has been implicated in the development of insulin resistance. We propose to test associations of muscle mass with insulin sensitivity and their respective associations with animal and vegetable sources of protein intake, independently from adiposity. PARTICIPANTS: Non-diabetic participants aged 68-82 years from the NuAge study with all available measures (n=441) were included. MEASUREMENTS: A model considering age, sex, chronic diseases, physical activity; smoking and sources of protein intake influencing body composition components and insulin sensitivity was created and tested with Path Analysis for their independent associations. Muscle mass index (MMI; kg/height in m2) and % body fat were derived from DXA and BIA. Insulin resistance was estimated by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) score and physical activity by the Physical Activity Scale for the Elderly (PASE) questionnaire. Protein intakes were obtained from three non-consecutive 24h-diet recalls. RESULTS: In the final model, direct positive associations were observed between HOMA-IR score and MMI (ß=0.42; 95%CI: 0.24; 0.6) and % body fat (ß=0.094; 95%CI: 0.07; 0.11). There were no direct associations between animal protein intake and MMI or with HOMA-IR. There was a significant direct negative association between plant protein intake and MMI (ß= -0.068; 95%CI: -0.13; -0.003) and significant indirect associations mediated through MMI and % body fat between HOMA-IR and animal protein intake (ß=0.0321; 95%CI: 0.01; 0.05), as well as plant protein intake (ß= -0.07; 95%CI: -0.1; 0.0). CONCLUSIONS: Our final model indicated that MMI and HOMA score were significantly positively associated. Protein intake sources were related to HOMA-IR score differently through MMI and % body fat, respectively.

Regulation of the pituitary growth hormone-releasing hormone receptor in ageing male and female LOU rats: new insights into healthy ageing.

Ageing is characterised by a decrease of somatotroph functionality, involving growth hormone-releasing hormone receptor (GHRH-R). The present study was conducted in LOU/C/jall (LOU) rats, a strain described as a model of healthy ageing, which is characterised by a low adiposity and long life expectancy without developing severe pathologies. Effects of age and diet (chow versus self-selection), on levels of anterior pituitary GHRH-R mRNA transcripts, were assessed in male and female LOU rats. The effect of age on pituitary GHRH-R functionality was examined in the anterior pituitary of both males and females fed chow diet. Moreover, serum insulin-like growth factor-I (IGF-I), T4 and leptin were measured because changes in their concentration could affect GHRH-R expression. In the pituitary of 18-month-old male and female LOU/C/jall rats fed standard chow, the level of 2.5-kb GHRH-R mRNA transcript, coding for functional GHRH-R, was significantly decreased. In 24- to 34-month-old males and females, it progressively returned to the level of younger animals, suggesting an enrichment of the group with survivors maintaining functional GHRH-R. In males and females repeatedly submitted to self-selection, this phenomenon was not observed. Studies with the GHRH-R agonist, Fluo-GHRH, revealed that 73% of 16-18-month-old male and female rats studied did not show an increase of fluorescence density characteristic of receptor-mediated internalisation upon incubation at 37 degrees C. In the other 27%, the increase of fluorescence was identical to that observed in pituitaries of young rats, suggesting the presence of an optimal level of functional GHRH-R. Serum levels of leptin, free T4 and total IGF-I decreased more drastically in ageing males and in rats fed a self-selection diet. A positive correlation was demonstrated between leptin and IGF-I levels in ageing males and females fed standard chow and ageing females submitted to a self-selection regimen. In conclusion, healthy ageing in LOU rats fed chow diet appears to be associated with a maintenance of functional pituitary GHRH-R levels found in younger rats but not necessarily with those of serum leptin, T4 and IGF-I.

Cellular distribution of growth hormone-releasing hormone receptor in human reproductive system and breast and prostate cancers.

Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreover, GHRH-R was demonstrated in prostate and breast carcinomas, opening a variety of possibilities for the use of GHRH antagonists in the treatment of prostatic and mammary tumors.

Dietary patterns and incidence of depression in a cohort of community-dwelling older Canadians.

OBJECTIVES: To investigate the association of dietary patterns with a 3-year incidence of depression among healthy older adults. DESIGN: Multiple logistic regression models adjusted for age, sex, marital status, smoking, education, total energy intake, physical activity, body mass index, hypertension, functional autonomy, cognitive functioning, social activities, and stressful life events. Energy and macronutrient intakes were also analyzed as potential predictors of depression. SETTING: Cities of Montréal, Laval, and Sherbrooke in Quebec, CA. PARTICIPANTS: Community-dwelling older adults, free of depression at baseline (N=1,358, 67-84 y), followed for 3y in the Québec Longitudinal Study on Nutrition and Aging (NuAge). MEASUREMENTS: Dietary patterns derived from principal components analysis of three 24 h-recalls at baseline, and depression incidence as measured by the 30-item Geriatric Depression Scale (≥11) and/or use of antidepressants at follow-up years. RESULTS: 170 people (63% women) became depressed over the 3 years. People in the highest tertile of adherence to the "varied diet" had lower risk of depression before adjustment (OR 0.58, 98% C.I. 0.38-0.86) but not significant once age and sex were controlled. No other dietary pattern was associated with the incidence of depression. The highest tertile of energy intake was associated with lower depression incidence after controlling for all confounders (OR 0.55, 95%CI 0.34-0.87). CONCLUSION: Among healthy older adults, dietary patterns do not appear to be related to depression. Those who eat less, however, possibly reflecting declining health, are at higher risk of becoming depressed.

Strategies in neuropeptide receptor binding research.

Strategies and general approaches used in neuropeptide receptor binding assays are described. Special attention is given to the nature of the ligand, its physical and chemical stability and the demonstration of an appropriate ligand selectivity pattern. Examples are given to illustrate critical aspects of neuropeptide receptor binding assays. Strong correlation between binding and bioassay data is also stressed.

Alterations of pituitary growth hormone-releasing factor binding sites in aging rats.

This study was designed to determine whether growth hormone (GH)-releasing factor (GRF) binding sites are altered in parallel to the diminution of GH secretion that occurs in aging. Using [125I-Tyr10] hGRF (1-44)NH2 as radioligand, we performed cold saturation studies in 2, 8, 14 and 18-month-old Sprague-Dawley male rat pituitary homogenates. In young rats (2 months), analysis by Ligand revealed the presence of two distinct classes of binding sites (KDs = 0.86 +/- 0.15 and 400 +/- 210 nM; BMAXS = 269 +/- 47 fmol and 42 +/- 19 pmol/mg protein, respectively). In 8 and 14-month old rats, there was a concomitant decrease in capacity of the high affinity class of sites (P less than 0.01) and increase in capacity of the low affinity class of sites (P less than 0.05). In parallel, a transient increase in affinity of the high affinity class of sites was observed in 14-month-old rats (P less than 0.05). In old rats (18 months), the data were no longer statistically analyzed with a two site-model, indicating a severe blunting of the high affinity sites. As the GRF-induced GH secretion is still not diminished at 8 month of age in these animals, our results indicate: 1) that alterations of GRF binding sites precede the GH impairment, thus probably participate in the initiating of this phenomenon and 2) that the biological actions of GRF on GH secretion are likely mediated by the high affinity class of sites.

Differential in vivo regulation of the pituitary growth hormone-releasing hormone (GHRH) receptor by GHRH in young and aged rats.

In aging, alterations of pituitary GH-releasing hormone (GHRH) receptor (GHRH-R)-binding sites have been proposed as one of the initiating factors contributing to the loss of somatotroph responsiveness to GHRH. Changes in the characteristics and/or concentration of the functional GHRH-R could take place in the course of aging and reduce the sensitivity of the somatotroph axis to GHRH. Because chronic exposure to GHRH has been proposed to resensitize aged somatotroph cells, better knowledge of its effects on the regulation of the somatotroph axis is required, particularly at the level of GHRH-R. Two- and 18-month-old male Sprague Dawley rats were treated for 14 days with a daily s.c. injection of 0.5 or 1.0 mg/kg BW human GHRH-(1-29)NH2 or saline. In 2-month-old rats, treatment with 0.5 mg/kg GHRH increased the number of high affinity pituitary GHRH-R-binding sites by 2-fold (P < 0.05) and hypothalamic somatostatin (SRIF) content by 45% (P < 0.05). It did not affect hypothalamic GHRH content, serum total insulin-like growth factor I (IGF-I), or body weight gain. Treatment with 1.0 mg/kg GHRH decreased the number of high affinity pituitary GHRH-R-binding sites by 2.4-fold compared with that in rats treated with 0.5 mg/kg BW (P < 0.05) and increased hypothalamic SRIF content by 45% (P < 0.05), but did not affect GHRH content. It also decreased circulating levels of IGF-I by 13% (P < 0.05) and slowed the growth rate by 17% (P < 0.05). In 18-month-old rats, treatment with 0.5 mg/kg GHRH for 14 days was not sufficient to rejuvenate pituitary GHRH binding parameters. However, treatment with 1.0 mg/kg GHRH restored the affinities of high and low affinity classes of GHRH-binding sites to values similar to those found in 2-month-old rats. Binding capacities of the high and low affinity classes of sites were increased by 1.8- and 3-fold, respectively, although significance was only reached for the low affinity site (P < 0.05). These changes were associated with a normalization of the level of 2.5-kb GHRH-R messenger RNA transcript, which was decreased by 31% in aging rats (P < 0.05), and by a trend for an increase in the 4-kb GHRH-R messenger RNA transcript, which was already increased by 49% in 18-month-old rats (P < 0.05). A normalization of serum IGF-I levels, which were decreased by 11% in 18-month-old control rats (P < 0.01), was also observed. No treatment effect was detected on body weight or hypothalamic SRIF and GHRH contents. We conclude that a 14-day administration of GHRH induces a differential GHRH-R-mediated regulation at the level of the pituitary and probably the hypothalamus as a function of age.

Effects of active immunization against somatostatin on serum growth hormone concentration in growing pigs: influence of fasting and repetitive somatocrinin injections.

Three experiments were conducted with growing pigs actively immunized against a protein-conjugated somatostatin (SRIF) in Freund's adjuvant. In the first experiment, blood from 24-week-old pigs (seven immunized and eight control) was sampled at 20-min intervals for 6 h to evaluate basal GH concentrations. The animals were then injected iv with porcine GH-releasing factor (GRF)-(1-29)NH2 (10 micrograms/kg). Before GRF stimulation, immunized animals had higher (P less than 0.05) baseline mean GH levels (2.6 vs. 1.4 ng/ml) and area under the GH curve (AUC; 1632 vs. 779 ng/min.ml); they also had higher AUC after GRF administration (4268 vs. 1972 ng/min.ml). In a second experiment eight immunized and eight control pigs were injected iv four times at 90-min intervals with porcine GRF (10 micrograms/kg). Control pigs responding to the first injection did not respond to the second and third, and those responding to the second did not respond to the first, third, and fourth, indicating a decreased responsiveness that was longer than 3 h post-GRF response in control pigs. SRIF-immunized pigs had a more consistent GH response to the GRF injections. Overall, a reduced response was observed after the second and the fourth injections in immunized pigs, although five and six of eight animals had a GH peak response higher than 10 ng/ml during these periods. In a third experiment, effects of fasting, GRF, and SRIF immunization were studied. Immunization and fasting had their own positive effects on serum GH levels. Immunization increased baseline mean GH levels (5.0 vs. 2.2 ng/ml) and total AUC before (2318 vs. 1073 ng/min.ml) and after (1886 vs. 910 ng/min.ml) iv GRF stimulation (10 micrograms/kg) compared to controls. Fasting increased the mean baseline GH level (4.5 vs. 2.6 ng/ml), and it increased AUC before exogenous GRF stimulation (2009 vs. 1392 ng/min.ml). In conclusion, SRIF in pigs seems to be a potent GH-governing factor, since, when inhibited, baseline mean GH levels increase, and a consistent response to GRF is observed. Fasting could increase GH concentrations by different ways: decreasing SRIF release and increasing GRF release or modifying the sensitivity of the somatotrophs to both factors.

Affinity of human growth hormone-releasing factor (1-29)NH2 analogues for GRF binding sites in rat adenopituitary.

Previous research on growth hormone-releasing factor analogues has used pituitary cell culture assay systems to evaluate in vitro their biological activity. However, binding assay systems in which receptor affinity and peptide stability can be assessed independently have been lacking so far. Since we have recently developed a sensitive GRF binding assay with [125I-Tyr10]hGRF(1-44)NH2, this method was applied to structure-affinity studies as a first step of screening GRF analogues. Acylation of the N-terminus of hGRF(1-29)NH2 generally decreased its affinity (relative affinity to hGRF(1-29)NH2 (RA), 26-85%). Replacement of the C-terminal carboxamide by a free carboxylic function decreased affinity likely by diminishing its proteolytic stability (RA, 57%). Removal of Tyr1, Ser9, Lys12, Val13, Gly15, Gln16, or Lys21 drastically decreased its affinity (RA, less than 3%). Multiple amino acid deletions in the segment 13-21 of hGRF(1-29)NH2 also led to a loss of affinity as did replacing segment 13-15, 16-18, or 19-21 by an octanoyl moiety (RA, less than 1%). Removal of Asn8, Gln24, Asp25, Ile26, Met27, and Ser28 or Arg29 had less effect on GRF receptor affinity (RA, 5-33%). Removal of Met27 or Ser28 only slightly affected hGRF(1-29)NH2 affinity (RA, 62-78%). Altogether, these results indicate that the amino acids contained in the segment 13-21 are more important than those of 24-29 to insure high affinity receptor binding or to maintain an optimal conformation to allow GRF binding.

Synthesis and inhibitory potency of peptides corresponding to the subunit 2 C-terminal region of herpes virus ribonucleotide reductases.

H-Tyr329-Ala330-Gly331-Ala332-Val333-Va l334-Asn335-Asp336-Leu337-OH, the C-terminal end of herpes simplex virus ribonucleotide reductase subunit 2 (HSV R2), specifically inhibits viral enzyme activity by interacting with subunit 1 (HSV R1). In a previous structure-activity study, we identified four sites on the nonapeptide where the inhibitory potency could be modulated: a minimum active core 333-337, a spacer segment 330-332, and the N- and C-termini. To further explore the structural features of HSV R2-(329-337) that are required to obtain a potent inhibition, a series of analogues comprising modifications in these four regions were synthesized by solid-phase methodology. Changes in the segment 333-337 of the molecule decreased the inhibitory potency by more than 2-fold, except for the Ile334 substitution, which resulted in a 1.5-fold increase in potency. Replacement of Tyr329 by other aromatic or aliphatic amino acids diminished the nonapeptide activity from 1.4-fold to 5.9-fold. The spacer segment contributed to enhance potency. Modification with amino acids that could induce conformational changes, such as Pro or D-Ala, generated compounds with a similar or lower activity, respectively. Amidation or amino acyl addition at the carboxylic end was detrimental while acylation of the N-terminus was generally beneficial for the inhibitory potency. Disubstitution in position 332 and 334 by Thr and Ile, which are present in the C-terminal portion of varicella-zoster virus ribonucleotide reductase subunit 2, resulted in a peptide that is 4.0 times more potent than HSV R2-(329-337), while each monosubstitution alone generated peptides with 150% of the activity of HSV R2-(329-337) nonapeptide. These results indicate a synergistic effect of the disubstitution which confers to this analogue physicochemical properties enhancing its ability to interact with its R1 binding site.

Structure-function studies of peptides inhibiting the ribonucleotide reductase activity of herpes simplex virus type I.

Ac-Tyr298-Ala299-Gly300-Thr301-Val302-I le303-Asn304-Asp305-Leu306-OH (Ac-VZV R2-(298-306)) represents the acetylated form of the C-terminus of varicella-zoster virus (VZV) ribonucleotide reductase subunit 2 (R2). This peptide possesses a high degree of homology with the C-terminus nonapeptide of the herpes simplex virus (HSV) type I and II ribonucleotide reductase R2 protein and is 15 times more potent than the latter in its in vitro inhibition of HSV-1 reductase activity. Accordingly, a new series of analogues based on this structure was studied in vitro. The replacement of Asp305 by Asn, Glu, Gln, Ser, or Cys; of Asn304 by Gln or Ser; of Ile303 and Val302 by D-Val; and of Tyr298 by Cha induced an important loss of inhibitory potency. The substitution of Asn304 by Asp; of Thr301 by Cys, Ser, or Val; of Gly300 by Ala or Val; of Ala299 by Val; or of Tyr298 by homoPhe, 4'-fluoro-Phe, 4'-chloro-Phe, 3'-iodo-Tyr, Me-Tyr, or For-Trp led to a moderate decrease of the Ac-VZV R2-(298-306) potency. The replacement of Val302 by Ile; Ala299 by Cys, Ser, or Thr; or the insertion of a six- or eight-carbon chain between Tyr298 and the NH2 terminus either preserved or slightly increased the inhibitory potency of Ac-VZV R2-(298-306). Finally, the substitution of Tyr298 by Trp or the addition of 4'-nitro-Phe at the amino terminus resulted in a 3-fold increase of potency. Altogether, these results stress the importance of the structural integrity of the minimum active core 302-306 in preserving the inhibitory potency and suggest that further studies on monosubstitutions could be directed at the portion 298-301 of the peptide.

Dopamine D1 receptor ligands modulate cognitive performance and hippocampal acetylcholine release in memory-impaired aged rats.

The possible modulation by D1 drugs of learning abilities of a population of aged memory-impaired animals was investigated in the present study. The level of D1/[3H]SCH 23390 receptors was first examined by quantitative autoradiography to ascertain if cognitive deficits seen in these animals could be related to alterations in the levels of these receptors. No significant differences in [3H]SCH 23390 binding were observed in any of the brain areas examined between young, and aged memory-unimpaired and aged memory-impaired animals. However, the cognitive deficits of the aged-impaired rats were modulated by D1 drugs. The D1 agonists SKF 38393 and SKF 81297 (3.0 mg/kg, i.p.) significantly reduced the latency period to find a hidden platform in the Morris Water Maze, reflecting improved cognitive functions, while the D1 antagonist SCH 23390 (0.05 mg/kg, i.p.) had no overall significant effect. Moreover, the D1 agonist SKF 38393 increased, whereas the antagonist inhibited, in vivo hippocampal acetylcholine release. Taken together, these results suggest that functional hippocampal acetylcholine-dopamine interactions exist in aged memory-impaired rats. More importantly, the cognitive deficits seen in the aged-impaired rats can be attenuated by stimulations of D1 receptors, hence suggesting an alternative approach to alleviate the cognitive deficits seen in the aged brain.

Similar ultrastructural distribution of the 5-HT(2A) serotonin receptor and microtubule-associated protein MAP1A in cortical dendrites of adult rat.

As visualized by light and electron microscopic immunocytochemistry, the distribution of the neuronal serotonin-2A (5-HT(2A)) receptor is mainly intracellular throughout adult rat brain. This localization is particularly striking in the pyramidal cells of cerebral cortex, the dendrites of which are intensely immunoreactive, but without any labeling of their spines. In view of recent yeast two-hybrid and biochemical results suggesting an association of 5-HT(2A) receptors with the cytoskeletal microtubule-associated protein MAP1A, the respective subcellular distributions of the receptors and of MAP1A were compared by quantitative electron microscopic immunocytochemistry in dendrites of adult rat frontoparietal cortex. Counts of silver-intensified immunogold particles revealed a higher density of 5-HT(2A) receptors in smaller rather than larger dendrites, and an apportionment between pre-defined compartments representing the plasma membrane and the cytoplasm that was proportional to the relative surface area of these compartments. MAP1A immunoreactivity also predominated in smaller versus larger dendrites, but with a slightly lower proportion of labeling in the plasma membrane versus cytoplasmic compartment. The co-localization of 5-HT(2A) receptors and MAP1A protein in the same dendrites could be demonstrated in double immunolabeling experiments. These results confirmed the predominantly somato-dendritic, intracellular localization of 5-HT(2A) receptors in cerebral cortex, showed their higher concentration in distal as opposed to proximal dendrites, and suggested their potential association to the cytoskeleton in cortical neurons in vivo. Such a distribution of 5-HT(2A) receptors reinforces our earlier hypothesis that 5-HT(2A) receptors participate in intraneuronal signaling processes involving the cytoskeleton, and raises the possibility that their activation could be dependent upon that of another co-localized, plasma membrane-bound, 5-HT receptor.