Enhanced retinoid response by a combination of the vitamin A ester retinyl propionate with niacinamide and a flavonoid containing Ceratonia siliqua extract in retinoid responsive in vitro models.

OBJECTIVES: Retinoids have been used for decades as efficacious topical agents to treat photoaged skin. The purpose of our present research is to evaluate whether the activity of the vitamin A ester retinyl propionate (RP) can be enhanced by niacinamide (Nam) and a flavonoid containing Ceratonia siliqua (CS) fruit extract in retinoid responsive in vitro models. METHODS: Retinyl propionate was tested alone and in combination with Nam and CS in an RARα reporter cell line for promoter activation and compared to trans-retinoic acid (tRA) activation. These treatments were also tested in keratinocytes for gene expression profiling by qPCR using a panel of 40 retinoid responsive genes. RESULTS: tRA or RP elicited RARα reporter activation in a dose-dependent manner. The combination of 0.5 µM or 2 µM RP with 10 mM Nam had a 56% and 95% signal increase compared to RP, respectively. The addition of 1% CS to 0.5 µM or 2 µM RP with 10 mM Nam elicited a further increase of 114% and 156%, respectively, over RP and Nam combinations. All retinoids elicited an increase in expression of 40 retinoid sensitive genes over control levels. Of the 40 genes, 27 were enhanced by either 0.1 µM RP or 0.5 µM RP with 10 mM Nam and 1% CS. Nam or CS had very modest activity in both models. CONCLUSION: The combination of RP with Nam and CS showed a higher retinoid response than RP in two separate retinoid responsive in vitro models. We hypothesize Nam and CS enhances RP activity by modulating metabolism to tRA via increasing NAD+ pools and inhibiting reduction of retinal (RAL) back to retinol, respectively. The findings provide evidence that this combination may have enhanced efficacy for treating the appearance of photoaged skin.

OBJECTIFS: Les rétinoïdes sont utilisés depuis des décennies comme agents topiques efficaces pour traiter la peau photo-âgée. Le but de notre recherche actuelle est d'évaluer si l'activité du propionate rétinyl ester de vitamine A (RP) peut être augmentée par le niacinamide (Nam) et un flavonoïde contenant un extrait de fruit de Ceratonia Siliqua (CS) dans les modèles in vitro sensibles aux rétinoïdes. MÉTHODES: RP a été testé seul et en combinaison avec Nam et CS dans une ligne de cellule rapporteur de RARα pour l'activation du promoteur et par rapport à l'activation de l'acide transrétinoïque(tRA). Ces traitements ont également été testés dans les kératinocytes pour le profilage d'expression génique par qPCR à l'aide d'un panel de 40 gènes rétinoïdes sensibles. RÉSULTATS: tRA ou RP ont provoqué l'activation du promoteur RARα d'une manière dépendante de la dose. La combinaison de 0,5 µM ou 2 µM de RP avec 10 mM de Nam a permis une augmentation respectivement de 56% et 95% du signal par rapport à RP. L'ajout de 1 % de CS à 0,5 µM ou 2 µM de RP avec 10 mM de Nam a permis une nouvelle augmentation de 114 % et 156 %, respectivement, qu'avec la combinaison RP et Nam. Tous les rétinoïdes ont provoqué une augmentation de l'expression de 40 gènes sensibles aux rétinoïdes sur les niveaux de contrôle. Sur les 40 gènes, 27 ont été améliorés soit par 0,1 µM de RP ou 0.5 µM de RP avec 10 mM de Nam et 1% de CS. Nam ou CS avaient une activité très modeste dans les deux modèles. CONCLUSION: La combinaison de RP avec Nam et CS a montré une réponse rétinoïde plus élevée que RP dans deux modèles in vitro séparés sensibles aux rétinoïdes. Nous émettons l'hypothèse que Nam et CS améliorent l'activité RP en modulant le métabolisme de tRA par l'augmentation des groupement NAD+ et en inhibant la réduction du rétinal (RAL) en rétinol, respectivement. Les résultats fournissent la preuve que cette combinaison peut améliorer l'efficacité du traitement de l'aspect de la peau photo-âgée.

Somatostatin receptors 2 and 5 are preferentially expressed in proliferating endothelium.

Angiogenesis is characterised by activation, migration and proliferation of endothelial cells and is central to the pathology of cancer, cardiovascular disease and chronic inflammation. Somatostatin is an inhibitory polypeptide that acts through five receptors (sst 1, 2, 3, 4, 5). Sst has previously been reported in endothelium, but their role remains obscure. Here, we report the expression of sst in human umbilical vein endothelial cells (HUVECs) in vitro, during proliferation and quiescence. A protocol for culturing proliferating and quiescent HUVECs was established, and verified by analysing cell cycle distribution in propidium-iodide-stained samples using flow cytometry. Sst mRNA was then quantified in nine proliferating and quiescent HUVEC lines using quantitative reverse transcriptase-polymerase chain reaction. Sst 2 and 5 were preferentially expressed in proliferating HUVECs. All samples were negative for sst 4. Sst 1 and 3 expression and cell cycle progression were unrelated. Immunostaining for sst 2 and 5 showed positivity in proliferating but not quiescent cells, confirming sst 2 and 5 protein expression. Inhibition of proliferating cells with somatostatin analogues Octreotide and SOM230, which have sst 5 activity, was found (Octreotide 10(-10)-10(-6) M: 48.5-70.2% inhibition; SOM230 10(-9)-10(-6) M: 44.9-65.4% inhibition) in a dose-dependent manner, suggesting that sst 5 may have functional activity in proliferation. Dynamic changes in sst 2 and 5 expression during the cell cycle and the inhibition of proliferation with specific analogues suggest that these receptors may have a role in angiogenesis.

The antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells.

When cultured insect cells (Sf9) were grown in the presence of 5 x 10(-6) M azadirachtin, there was a rapid increase in the mitotic index, with the appearance of many aberrant mitotic figures. Flow cytometry established that cells accumulated in the G2/M phase of the cell cycle, and that the effect was concentration-dependent. At 10(-8) M a period of 20 h was necessary to raise the proportion in G2/M to 42% above the control values, but at 5 x 10(-6) M more than 90% of the cells were in this phase. Azadirachtin had the same effect on C6/36 mosquito cells, but failed to affect L929 murine fibroblast cells even at a concentration of 10(-4) M over 72 h. Experiments with colchcine and taxol showed similarities of action between azadirachtin and colchicine, and azadirachtin was apparently able to displace colchicine-fluorescein from binding-sites in living insect cells. Another similarity between azdirachtin and colchicine was that both phytochemicals prevented the polymerisatrion in vitro of mammalian tubulin, although the azadirachtin was much less effective.

Two-Dimensional detection in ion chromatography: sequential conductometry after suppression and passive hydroxide introduction.

An improved method that uses sequential suppressed and nonsuppressed IC for the sensitive detection of both common anions and very weak acid anions is described. After suppressed conductometric detection of an electrolytically generated hydroxide eluent and an electrolytic suppressor, the eluent is passed into a membrane device where KOH is passively introduced into the eluent stream using Donnan forbidden leakage. A second conductivity detector then measures the conductivity of the stream. The background conductance of the second detector is typically maintained at a relatively low level of 20-30 microS/ cm. The weak acids are converted to potassium salts that are fully ionized and are detected against a low KOH background as negative peaks. The applicability of different commercially available cation exchange membranes was studied. Device configurations investigated include planar, tubular, and a filament-filled annular helical (FFAH) device. The FFAH device provided more effective mixing of the penetrated hydroxide with the eluent stream, resulting in a noise level of < or = 7 nS/cm and a band dispersion value of less than 82 microL. Optimal design and performance data are presented.

Flavor-protein binding: disulfide interchange reactions between ovalbumin and volatile disulfides.

The irreversible binding of selected sulfur-containing flavor compounds to proteins was investigated in aqueous solutions containing ovalbumin and a mixture of disulfides (diethyl, dipropyl, dibutyl, diallyl, and 2-furfuryl methyl) using solid-phase micro-extraction (SPME). In systems which had not been heated, the recovery of disulfides from the headspace above the protein at the native pH (6.7) was similar to that from an aqueous blank. However, significant losses were observed when the pH of the solution was increased to 8.0. When the protein was denatured by heating, much greater losses were observed and some free thiols were produced. In similar heat-denatured systems at pH 2.0, no losses of disulfides were observed. Disulfides containing allyl or furfuryl groups were more reactive than saturated alkyl disulfides. Interchange reactions between protein sulfhydryl groups and the disulfides are believed to be responsible for the loss of the disulfides.

In vitro methylation of predetermined regions in recombinant DNA constructs.

DNA methylation at position 5 in the cytosine ring in the sequence CpG can be detrimental to the transcription of a variety of genes in higher eukaryotes (1,2). Although the significance of this transcriptional repression is currently under debate (3,4), there is little disagreement that it plays an important role in genomic imprinting and X-chromosome inactivation (5,6). To study the effects of DNA methylation on transcription in an experimental system, bacterial DNA methyltransferases have been used widely in order to mimic the DNA methylation pattern of eukaryotic genes. However, usually every target site in a given recombinant DNA molecule will be subject to DNA methylation by making use of those enzymes. This might result in an exaggeration of the effects of DNA methylation, as most recombinant DNA molecules contain a high degree of prokaryotic DNA, which is rich in CpGs. This methylated CpGrich DNA can contribute to the effects of DNA methylation by formation of a repressive chromatin structure (7,8). In addition, selective DNA methylation is required to distinguish the effects of DNA methylation on transcription initiation and transcript elongation (8,9). Thus, there is a requirement for a method to generate recombinant DNA molecules that are methylated in a predetermined region. The chapter following this one will describe a method that makes use of ligation of methylated DNA fragments into unmethylated vector DNA. This method relies on the availability of suitable restriction sites, which allow directional cloning of the fragment and, in addition, requires a highly efficient ligation reaction.

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat.

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.

Prediction of Boston Naming Test performance from vocabulary scores: preliminary guidelines for interpretation.

Patients with limited education or underdeveloped vocabulary skills may perform below the normal range on the Boston Naming Test when compared to the original published norms, even in the absence of brain damage. To reduce the frequency of false positive dysnomic classifications of patients with limited vocabulary skills, we developed a score adjustment to account for the significant shared variance between scores on this test and the WAIS-R Vocabulary subtest. Vocabulary significantly predicted performance on the Boston Naming Test (r = .65, p < .0001) in a sample of 62 outpatients who had no objective evidence of brain damage. Linear regression was used to derive expected performance on the Boston Naming Test from Vocabulary scaled scores. Relative to the original published norms, scores based on the Vocabulary subtest cut-offs produced fewer false positives and more accurately classified group membership for patients with and without objectively verified brain damage. These performance predictions are offered as tentative guidelines to assist clinicians in evaluating the presence of naming deficits by controlling for the variance associated with knowledge of vocabulary.

Knee manipulation after total knee arthroplasty.

To determine if any factors are associated with knee stiffness after total knee arthroplasty (TKA), we retrospectively reviewed the medical records and radiographs of patients who had knee manipulation after total knee replacement at Scott & White Memorial Hospital from 1983 to 1993. Twenty-five patients who had knee manipulation after TKA were matched by surgeon, year of surgery, and age (+/- 5 years) with a study group of 25 patients who did not have knee manipulation after TKA. Patients in the manipulated group had decreased flexion at the time of discharge from the hospital after the knee arthroplasty and a decreased final flexion. The age of the patient, time from surgery to manipulation, and preoperative flexion did not correlate with final flexion attained in the manipulated group. Relative to the control study group, the manipulated group had an increase in postoperative anteroposterior femoral thickness. A decrease in patellar height was noted both in the manipulated group and in the control nonmanipulated group. There was no significant difference between groups for a change in patellar height.

Stanniocalcin 2: characterization of the protein and its localization to human pancreatic alpha cells.

Stanniocalcin (STC) is a hormone that was originally identified in fish, where it inhibits calcium uptake by the gills and gut and stimulates phosphate adsorption by the kidney. Recently, two mammalian homologues of stanniocalcin were identified. The first (STC1) shows 61% identity to the fish stanniocalcins and appears to have a function similar to that of the fish stanniocalcins. The second homologue (STC2) is 30-38% identical to the fish stanniocalcins, and is characterized by unique cysteine and histidine motifs that are not found in the other stanniocalcins. We purified both the native hamster and recombinant human STC2 proteins and obtained a partial amino acid sequence of the hamster protein. Both proteins behave as a disulfide bonded homodimer, which undergoes post-translational modification(s). The STC2 gene was localized to human chromosome 5q35. Northern blot analysis revealed that the primary site of human STC2 production is the pancreas, and immunostaining localized the STC2 protein to a subpopulation of cells in the islet. Double immunostaining for STC2 and either insulin or glucagon revealed that STC2 protein is found in the alpha cells, but not the beta cells. We speculate that STC2 may play a role in glucose homeostasis.

Effect of CWG methylation on expression of plant genes.

The presence of two DNA methyltransferases in Pisum raises the possibility that they serve different functions. In vitro methylation of CWG sequences in the strong cauliflower mosaic virus 35S promoter had no effect on reporter gene expression. In contrast, in vitro methylation of CWG sequences in the relatively weak, CG-deficient Phaseolus vulgaris rbcS2 promoter inhibited transcription. Expression of both constructs was strongly inhibited by extensive CG methylation. A search of published plant promoter sequences revealed that the CG content of promoters is very variable, with some promoters having typical CG islands. In contrast, the distribution of CWG sequences is more even with little evidence for CWG islands.

Clinical utility of the Trail Making Test practice time.

The Trail Making Test (TMT) is one of the most frequently used measures in clinical neuropsychology. Data obtained from the TMT practice times were analyzed to determine their utility in predicting success and failure on the full version of the test and to allow establishment of criteria by which to judge administration or discontinuation of the full test. Results indicated that TMT practice times were useful in predicting successful completion of Part A and B of the TMT. Tables are provided which describe the classification accuracy of various TMT practice times. These tables allow clinicians to select a practice-time cutoff and then use the cutoff as a heuristic to assist in the decision to administer the remainder of that particular part of the TMT or discontinue the test. A 20-s cutoff resulted in optimal prediction of successful completion (< 180 s) of TMT Part A. A cutoff of 30 s optimally predicted successful completion (< 300 s) of TMT Part B.

Efficiency of expression of transfected genes depends on the cell cycle.

Lipofection, a lipid-mediated DNA transfection procedure, was used to transfect synchronized L929 mouse fibroblast cells with a reporter plasmid containing the bacterial chloramphenicol acetyltransferase gene. The efficiency of gene expression was investigated on transfection of cells at different stages of the cell cycle. Our data show that expression of the reporter gene was minimal when transfection was performed in G0-phase and parallel experimental data disproved the possibility that the reduced expression observed was due to differential uptake at different times in the cell cycle. Investigation into the condensation state of the plasmid has shown that the low chloramphenicol acetyltransferase gene expression could be a direct consequence of the packaging of the plasmid into condensed chromatin when transfection occurs in G0-phase. The inactivation of the reporter gene is not reversed by growth of the cells in high serum or by treatment with Trichostatin A, a specific inhibitor of histone deacetylase, suggesting that the inactive chromatin formed in G0-phase cells lacks associated histone acetylase activity. In contrast, the high activity seen when cells in S-phase are transfected is enhanced even further by treatment with Trichostatin A.

Normative corrections for the Halstead Reitan Neuropsychological Battery.

This article investigates the adequacy of the Heaton-Grant-Matthews norms in correcting age and education effects in the Halstead Reitan Neuropsychological Battery. Two hundred and ninety neurological and 346 psychiatric patients were administered the Halstead Reitan Neuropsychological Battery. Raw scores were converted to standard scores using the Heaton-Grant-Matthews norms. Ten percent of the variance in standard score profiles could be predicted by patient age and education. The primary effect of age was to decrease the overall level of the profile; the primary effect of education was to increase relative scores on the Aphasia test. When these standard scores were converted to age- and education-corrected T-scores, less than 1% of the variance could be predicted by age and education. When individual tests were examined, the Heaton-Grant-Matthews norms failed to correct 2% of the variance in the Speech Sounds test. Discussion focuses on the usefulness of the Heaton-Grant-Matthews norms in interpreting performance on the Halstead Reitan Neuropsychological Battery.

Photographic standards in plastic surgery.

Standard photographic technique in plastic surgery is an important topic that has been stressed in the discipline over the past several years. Clinical photographs should always be taken with the same camera lens, lens setting, lighting, film, and patient position to ensure reproducibility and to enable valid pre- and postoperative comparisons. A 35-mm single lens reflex camera is highly recommended for this type of photography. Two lenses are suggested, one with a focal length range of 50 to 60 mm and one with a focal length range of 90 to 105 mm. Both should have macro capability. Two or more flash units are recommended, either camera-mounted or a studio system set-up in the office. Using the patient preparation method and technique outlined in the text, the Standards in Clinical Photography achieve consistency from patient to patient and also in the same patient in pre- and postoperative photographs. Henceforth, the information discussed in the article forms the basis for standard views, regardless of the image-capture medium.

Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum.

A series of overlapping clones complementary to the Arabidopsis cytosine-5 DNA methyltransferase (C-5 MTase) has been isolated from pea cDNA libraries. The assembled nucleic acid sequence contains an open reading frame of 4761 bp encoding a protein of 1554 amino acids. Like other eukaryotic C-5 MTases, the inferred protein has a presumed regulatory N-terminal region linked to a catalytic C-terminal domain, which has eight of the ten conserved motifs found in prokaryotic C-5 MTases. The pea C-5 MTase has 65% identity at the nucleotide level and 61% identity at the protein level, with the Arabidopsis C-5 MTase. The catalytic domain of the pea enzyme shares 78% identity with Arabidopsis and approximately 52% identity with murine and human C-5 MTases, including the relative position of the proline-cysteine dipeptides of the catalytic centre. Using the conserved region of the cDNA as a probe, we have identified a transcript of 5 kb. Southern blot analysis of pea genomic DNA with the above probe indicates the presence of a single gene. Using poly(A)+ RNA from different developmental stages and different tissues, we have observed that expression is confined mostly to the rapidly dividing tissues of the plant. Expression of this assembled cDNA in a baculovirus system gives a protein of approximately 174 kDa. The expressed protein can be cross-linked, in an AdoMet-dependent manner, to duplex oligonucleotide substrates containing FdC in place of target cytosines in either CG or CAG/CTG sequences.

Comparison of two computational formulas for a WAIS-R seven subtest short form.

Several validity studies for a seven subtest WAIS-R short form have been conducted with patients from different populations as participants. All of these studies demonstrated high correlations between the short form IQ estimates and the actual VIQs, PIQs, and FSIQs (i.e., .90 to .98). In general, there also were small mean differences in the short form versus actual IQs across samples. There currently are two computational formulas for the seven subtest short form. The original weighted formula and a revised proration formula. This study investigated the accuracy of the two short form computational formulas in samples of patients with brain impairment. It was found that the two formulas produced nearly identical results from both statistical and clinical perspectives. Given that the formulas produce nearly identical results and the majority of published studies with this short form have used the weighted formula, we recommend that future investigators use the original weighted formula to maintain clinical and scientific consistency.

Laser-photosensitizer assisted immunotherapy: a novel modality for cancer treatment.

Photosensitizer-enhanced laser treatment, where dyes are activated in situ by lasers of appropriate wavelengths, provides highly selective tissue destruction, both spatially and temporally, through photophysical reactions. Although laser-sensitizer treatment for cancer can achieve a controlled local tumor cell destruction on a large scale, total tumor eradication may not be accomplished because of the incomplete local tumor killing or the presence of tumor metastases, or both. The long-term control of cancer depends on the host immune surveillance and defense systems in which both cell-mediated and humoral responses are critical. In this study we report a novel minimally invasive cancer treatment combining the laser photophysical effects with the photobiological effects. Irradiation of a rat mammary tumor by an 805 nm diode laser, after an intratumor administration of a specific photosensitizer, indocyanine green in a glycated chitosan gel, caused immediate photothermal destruction of neoplastic cells. Concomitantly this treatment stimulated the immunological defense system against residual and metastatic tumor cells. Increases in survival rate and in the eradication of tumor burden, both primary and metastatic, were observed after this treatment. Furthermore, the resistance of successfully treated rats to tumor rechallenge demonstrated a long-lasting systemic effect of the treatment. These findings indicate that our treatment has triggered a specific humoral immune response in the tumor-bearing rats.

Neuropsychological information in the Wechsler Adult Intelligence Scale-Revised.

The relationships among Wechsler Adult Intelligence Scale-Revised profiles (WAIS-R), Luria-Nebraska Neuropsychological Battery (LNNB) profiles and Halstead Retian Neuropsychological Battery (HRNB) profiles were examined in two samples of patients referred for neuropsychological evaluation. Canonical correlation analysis suggested that the average level of WAIS-R profiles was related to the average level and scatter of LNNB profiles, Overall performance on the HRNB was less strongly related to overall performance on the WAIS-R than was the LNNB. Patients who were similar to a WAIS-R modal profile characterized by relative deficits on performance subtests were more likely to be similar to LNNB modal profiles characterized by relative impairments on sensorimotor subtests. Patients who were similar to a WAIS-R modal profile characterized by relative deficits on verbal subtests were more likely to be similar to LNNB modal profiles characterized by relative impairments on either language subtests or conceptual subtests. Patients classified into an HRNB profile type characterized by strengths on the Aphasia Screening subtest were more likely to show strengths on WAIS-R verbal subtests. However, less than 8 % of the total samples could be jointly classified into both the requisite WAIS-R profile clusters and one of the associated LNNB or HRNB profile clusters. WAIS-R subtest profile level may be a useful statistic to screen for neuropsychological deficits, but WAIS-R patterns are essentially useless for neuropsychological screening. Discussion focuses on the role of the WAIS-R in neuropsychological evaluations.

Cross-validation of predicted Wechsler Memory Scale-Revised scores in a normative sample of 25- to 34-year-old patients.

Equations for prorating Wechsler Memory Scale-Revised General Memory (GM) and Delayed Recall (DR) index scores ([Woodard and Axelrod, 1995]) were confirmed in the [Mittenberg et al. (1992)] normative WMS-R sample of 50 subjects between the ages of 25 and 34, and confirmed in a separate clinical sample of 30 patients with closed head injury who were age, education, and gender matched with 30 subjects from that normative sample. Predicted GM and DR index scores fell within 6 points of the obtained scores for 98% of the [Mittenberg et al. (1992)] sample and 93% to 100% of the matched head injury and normative samples, despite statistically significant differences between these groups in obtained GM, DR, and percent retention of LM I and II and VR I and II. Six points is well within the standard error of measurement of these index scores. These findings confirm the earlier cross-validation results reported by [Axelrod et al. (1996)] in a mixed sample of traumatic brain injury and other neurological insult, and suggest that this method of estimating weighted score sums for WMS-R General Memory and Delayed Recall indices may be safely used in normative samples of patients in this age range as well as neurologically compromised patients without significantly impacting index score accuracy.