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Background: Regorafenib is a salvage option for metastatic colorectal cancer (mCRC). Understanding the prognostic factors for mCRC patients undergoing regorafenib treatment can help optimize therapeutic strategies. Materials and methods: We searched Taiwan’s National Health Insurance database for patients who began regorafenib treatment for mCRC between 1 September 2015 and 31 December 2018. Taiwan’s National Death Registry and the Taiwan Cancer Registry were examined for data on survival and clinicopathological variables, respectively. Results: In total, 3643 patients were included in the analysis. The median time to treatment discontinuation (TTD) was 2.3 months, and the median overall survival (OS) was 7.2 months. Compared with the patients with a right-sided tumor, those with a left-sided primary tumor exhibited a significantly longer TTD (2.4 versus 2.1 months, P < 0.001) and OS (7.6 versus 6.1 months, P < 0.001). The patients who received chemotherapy with regorafenib also exhibited a longer TTD (2.6 versus 2.2 months, P < 0.001) and OS (8.3 versus 6.7 months, P < 0.001) than did the patients who did not. In multivariate analysis, left-sidedness and chemotherapy addition were confirmed as predictors of longer TTD and OS. Because of the interaction between sidedness and KRAS mutation, we established separate Cox models and identified that left-sidedness was an independent predictor of longer TTD and OS for KRAS wild-type tumors but not for KRAS-mutant tumors. Conclusions: In this population-wide cohort study, left-sidedness of the primary tumor and the addition of chemotherapy were associated with a longer OS and TTD for regorafenib treatment for mCRC.

Background: The search for prognostic biomarkers indicating sensitivity to immunotherapy in lung adenocarcinoma patients has zeroed in on genes in the switch/sucrose non-fermentable (SWI/SNF) pathway. The mutational profiles of key genes are not clearly defined, however, and no comparisons have been conducted on whether mutations in the genes involved provide the same predictive value., Methods: In this study, analysis of clinical factors, tumor mutation burden (TMB), chromosomal instability, and co-alterations was conducted for 4344 lung adenocarcinoma samples. Independent online cohorts (N = 1661 and 576) were used to supplement the analysis with survival and RNA-seq data., Results: Mutational burden and chromosomal instability analysis showed that ARID family mutations (including ARID1A, ARID1B, or ARID2 mutations) and SMARC family mutations (including SMARCA4 or SMARCB1 mutations) display different profiles from wild-type (WT) samples (TMB: ARID versus WT: P < 2.2 × 10 -16 , SMARC versus WT: P < 2.2 × 10 -16 ; CIN: ARID versus WT: P = 1.8 × 10 -5 , SMARC versus WT: P = 0.027). Both mutant groups have a higher proportion of transversions than transitions, whereas the ratio is more equal for wild-type samples. Survival analysis shows that patients with ARID mutations were more sensitive to immunotherapy treatment than wild-type and SMARC-mutated patients (P < 0.001 and P = 0.013, respectively), and multivariate Cox analysis reveals that the presence of ARID mutations is likely the main cause., Conclusions: The research presented in this study shows that mutations in the ARID gene family, including ARID1A, ARID1B, and ARID2, are primarily responsible for the sensitive response to immunotherapy treatment in patients with lung adenocarcinoma., Competing Interests: Disclosure HT, HB, XW, YS are employees of Nanjing Geneseeq Technology Inc. All other authors have declared no conflicts of interest. Data Sharing The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. The data retrieved for the online validation section can be found on www.cbioportal.org and GDC Data Portal (https://portal.gdc.cancer.gov/)., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: We evaluated the non-inferiority of nedaplatin-based and cisplatin-based concurrent chemoradiotherapy in cervical cancer patients., Design: Patients aged 28-82 years with pathologically diagnosed cervical cancer (stage IB-IVA) were randomly chosen for the study. Patients in both the cisplatin and nedaplatin groups received radiotherapy and weekly intravenous nedaplatin 30 mg/m 2 or cisplatin 40 mg/m 2 concurrently., Results: One hundred and sixty patients who received treatment between 10 May 2018 and 31 August 2020 were included. The 3-year overall survival in the nedaplatin group (median 30.5 months) was not significantly different from that in the cisplatin group (28.5 months; hazard ratio 0.131, 95% confidence interval 0.016-1.068; P = 0.058). No significant differences in hematological toxicity were observed between the two groups. Vomiting (40 versus 61), nausea (44 versus 67), and anorexia (52 versus 71) were more common in the cisplatin group whereas effects on liver function, including total bilirubin (7 versus 3), alanine aminotransferase (7 versus 2), and aspartate aminotransferase (6 versus 2), were more common in the nedaplatin group. Four patients in the cisplatin group had grade I creatinine elevation, whereas none in the nedaplatin group had abnormal creatinine levels. Two patients in the nedaplatin group discontinued concurrent chemotherapy because of infusion, and one patient in the cisplatin group discontinued treatment because of infusion-induced dizziness., Conclusions: Our findings suggest that nedaplatin has a milder gastrointestinal reaction but a more significant effect on liver function than cisplatin. In patients with cervical cancer, nedaplatin-based concurrent chemoradiotherapy could serve as an alternative treatment to cisplatin., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Sequential treatment with different generations of anaplastic lymphoma kinase (ALK) inhibitors have been widely applied to ALK-positive lung cancer; however, resistance mutations inevitably developed. Further characterization of ALK resistance mutations may provide key guidance to subsequent therapies. Here we explored the emergence of secondary ALK mutations during sequential ALK tyrosine kinase inhibitor (TKI) treatment in a real-world study of Chinese lung adenocarcinoma (ADC) patients., Methods: A clinical-genomic database was queried for lung ADC patients with at least one ALK inhibitor treatment and at least one plasma sample collected following ALK inhibitor treatment. Targeted genome profiling was performed with a 139-gene panel in baseline tumor tissue and serial plasma samples of patients., Results: A total of 116 patients met inclusion criteria. ALK G1202R was more common in patients with echinoderm microtubule-associated protein-like 4 (EML4)-ALK v3 fusion, whereas ALK L1196M was more common in v1. TP53 mutant patients were significantly associated with harboring multiple ALK resistance mutations (P = 0.03) and v3+/TP53 mutant patients had the highest rate of multiple ALK resistance mutations. The sequential use of ALK TKI led to an increased incidence of concurrent ALK mutations along the lines of therapies. Alectinib had a lower rate (9%) harboring ALK resistance mutation as first-line ALK TKI compared with crizotinib (36%). ALK compound mutations identified included ALK D1203N/L1196M, ALK G1202R/L1196M, and ALK G1202R/F1174C, which may be lorlatinib resistant. Using paired pretreatment and post-treatment samples, we identified several ALK-independent resistance-related genetic alterations, including PTPRD and CNKN2A/B loss, MYC, MYCN and KRAS amplification, and EGFR 19del ., Conclusions: Sequential postprogression plasma profiling revealed that increased lines of ALK inhibitors can accelerate the accumulation of ALK resistance mutations and may lead to treatment-refractory compound ALK mutations. The selection for optimal first-line TKI is very important to achieve a more efficacious long-term strategy and prevent the emergence of on-target resistance, which may provide guidance for clinical decision making., Competing Interests: Disclosure XC, RY, HB, JL, XW, and YS are employees of Nanjing Geneseeq Technology Inc. All other authors have declared no conflicts of interest., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) has become a promising surrogate for genomic profiling of central nervous system tumors. However, suboptimal ctDNA detection rates from CSF limit its clinical utility. Thus precise screening of suitable patients is needed to maximize the clinical benefit., Patients and Methods: Between February 2017 and December 2020, 66 newly diagnosed non-small-cell lung cancer (NSCLC) patients with brain parenchymal metastases were prospectively enrolled as a training cohort and 30 additional patients were enrolled as an external validation cohort. CSF samples and matched primary tumor tissues were collected before treatment and subjected to next-generation sequencing (NGS). The imageological characteristics of patients' brain tumors were evaluated by radiologists using enhanced magnetic resonance imaging images. The clinical and imageological characteristics were evaluated by complete subsets regression, Akaike information criteria, and Bayesian information criteria methods to establish the prediction model. A nomogram was then built for CSF ctDNA detection prediction., Results: The somatic mutation detection rate of genes covered by our targeted NGS panel was significantly lower in CSF ctDNA (59.09%) than tumor tissue (91.84%). The T size (diameter of the largest intracranial lesion) and LVD min (minimum lesion-ventricle distance for all intracranial lesions) were significantly associated with positive CSF ctDNA detection, and thus, were selected to establish the prediction model, which achieved an area under the ROC curve (AUC) of 0.819 and an accuracy of 0.800. The model's predictive ability was further validated in the independent external cohort (AUC of 0.772, accuracy of 0.767) and by internal cross-validation. The CSF ctDNA detection rate was significantly improved from 58.18% (32/55) to 81.81% (27/33) in patients after model selection (P = 0.022)., Conclusions: This study developed a regression model to predict the probability of detecting CSF ctDNA using the phenotypic characteristics of metastatic brain lesions in NSCLC patients, thus, maximizing the benefits of CSF liquid biopsies., Competing Interests: Disclosure YM and YS are employees of Nanjing Geneseeq Technology Inc. The other authors have no conflicts of interest to declare., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Aims: Lipoprotein lipase (LPL) is a candidate gene for obesity based on its role in triglyceride hydrolysis and the partitioning of fatty acids towards storage or oxidation. Whether dietary fatty acids modify LPL associated obesity risk is unknown., Methods and Results: We examined five single nucleotide polymorphisms (SNPs) (rs320, rs2083637, rs17411031, rs13702, rs2197089) for potential interaction with dietary fatty acids for obesity traits in 1171 participants (333 men and 838 women, aged 45-75 y) of the Boston Puerto Rican Health Study (BPRHS). In women, SNP rs320 interacted with dietary polyunsaturated fatty acids (PUFA) for body mass index (BMI) (P = 0.002) and waist circumference (WC) (P = 0.001) respectively. Higher intake of PUFA was associated with lower BMI and WC in homozygotes of the major allele (TT) (P = 0.01 and 0.005) but not in minor allele carriers (TG and GG). These interactions were replicated in an independent population, African American women of the Atherosclerosis Risk in Communities (ARIC) study (n = 1334)., Conclusion: Dietary PUFA modulated the association of LPL rs320 with obesity traits in two independent populations. These interactions may be relevant to the dietary management of obesity, particularly in women., (Copyright © 2014. Published by Elsevier B.V.)

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17β-estradiol (InsHPrlE). The relative expression of β-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of β-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

IGF-1 plays an important role in cardiovascular homeostasis, and plasma levels of IGF-1 correlate inversely with systolic function in heart failure. It is not known to what extent circulating IGF-1 secreted by the liver and local autocrine/paracrine IGF-1 expressed in the myocardium contribute to these beneficial effects on cardiac function and morphology. In the present study, we used a mouse model of liver-specific inducible deletion of the IGF-1 gene (LI-IGF-1 -/- mouse) in an attempt to evaluate the importance of circulating IGF-I on cardiac morphology and function under normal and pathological conditions, with an emphasis on its regulatory role in myocardial phosphocreatine metabolism. Echocardiography was performed in LI-IGF-1 -/- and control mice at rest and during dobutamine stress, both at baseline and post myocardial infarction (MI). High-energy phosphate metabolites were compared between LI-IGF-1 -/- and control mice at 4 weeks post MI. We found that LI-IGF-1 -/- mice had significantly greater left ventricular dimensions at baseline and showed a greater relative increase in cardiac dimensions, as well as deterioration of cardiac function, post MI. Myocardial creatine content was 17.9% lower in LI-IGF-1 -/- mice, whereas there was no detectable difference in high-energy nucleotides. These findings indicate an important role of circulating IGF-1 in preserving cardiac structure and function both in physiological settings and post MI., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

It has been well established that serotonin (5-hydroxytryptamine, 5-HT) plays a key role in neuro-endocrine-immune networks, mostly through its receptors and/or transporters. Although the presence of 5-HT receptor mRNAs in peripheral blood mononuclear cells (PBMCs) of rhesus monkeys has been reported, there is little information about serotonin transporter (SERT) expression by these cells. To examine SERT expression at the transcription and translation level, one-step RT-PCR, confocal microscopy and flow cytometry were used to detect SERT mRNA and protein expression by rhesus monkey PBMCs. It was found that SERT mRNA could be detected by RT-PCR from all of the rhesus macaque PBMC RNA samples and the nucleotide sequence of the amplicons was identical to the published SERT mRNA sequence. Low level SERT immunoreactivity was also demonstrated on the surface of rhesus PBMCs by confocal microscopy. Almost all lymphocytes and most monocytes were positive for SERT by flow cytometry. In the 2 rhesus macaques examined by multicolor flow cytometry, SERT(bright) cells were more than 84%, 94%, and 96% among CD20+, CD3+, and CD3+CD4+ lymphocytes respectively. These data demonstrate expression of SERT by rhesus macaque PBMCs, and indicate that rhesus macaques would be suitable models to test the in vivo immune regulatory effects of 5-HT or drugs targeting SERT.

To define the renal tissue changes in stone-forming patients with distal renal tubular acidosis (dRTA), we performed intra-operative papillary and cortical biopsies in five patients. The main abnormalities were plugging of inner medullary collecting ducts (IMCD) and Bellini ducts (BD) with deposits of calcium phosphate in the form of apatite; epithelial cell injury and loss was marked. Plugged ducts were surrounded by interstitial fibrosis, but the fibrosis was generalized, as well, and was a main feature of the histopathology even when plugging was not present. In contrast, common idiopathic calcium oxalate stone formers (SF) never manifest intra-tubule crystals or interstitial fibrosis. Patients with brushite (calcium monohydrogen phosphate) stones and those with cystine stones have many fewer IMCD and BD plugged with apatite (or cystine, in cystinuria), and interstitial fibrosis is limited to the regions around plugged ducts. Patients with dRTA often present a radiographic picture of nephrocalcinosis. Our direct surgical observations reveal that these may be surgically removable stones, especially in patients with well preserved renal function. In all, dRTA SF have a more diffuse papillary renal disease than other SF thus studied, and are also unusual for the degree of interstitial fibrosis.

Background: Despite previous studies, uncertainty has persisted about the role of thymidylate synthase (TS) and p53 status as markers of prognosis in colorectal cancer (CRC)., Patients and Methods: A total of 967 patients accrued to a large adjuvant trial in CRC were included in a prospectively planned molecular substudy, and of them, 59% had rectal cancer and about 90% received adjuvant chemotherapy (either systemically or randomly allocated to intraportal 5-fluorouracil infusion or both). TS and p53 status were determined, blinded to any clinical data, by immunohistochemistry using a validated polyclonal antibody or the DO-7 clone, respectively, and their relationships with overall survival were examined., Results: High TS expression was observed in 58% and overexpression of p53 in 60% of tumours. TS expression correlated with tumour stage, and p53 overexpression, with rectal cancers. There was no evidence that either marker was significantly associated with survival by either univariate (TS hazard ratio (HR) = 0.94, 95% CI 0.76-1.18 and P = 0.6 and p53 HR = 0.98, 95% CI 0.78-1.23 and P = 0.9) or multivariate analyses (TS HR = 0.99, 95% CI 0.79-1.25 and P = 0.9 and p53 HR = 0.98, 95% CI 0.78-1.23 and P = 0.8)., Conclusions: Neither TS nor p53 expression has significant prognostic value in the adjuvant setting of CRC.

We have biopsied the papillae of patients who have cystine stones asking if this stone type is associated with specific tissue changes. We studied seven cystine stone formers (SF) treated with percutaneous nephrolithotomy using digital video imaging of renal papillae for mapping and obtained papillary biopsies. Biopsies were analyzed by routine light and electron microscopy, infrared spectroscopy, electron diffraction, and micro-CT. Many ducts of Bellini (BD) had an enlarged ostium, and all such were plugged with cystine crystals, and had injured or absent lining cells with a surrounding interstitium that was inflamed to fibrotic. Crystal plugs often projected into the urinary space. Many inner medullary collecting ducts (IMCD) were dilated with or without crystal plugging. Apatite crystals were identified in the lumens of loops of Henle and IMCD. Abundance of interstitial Randall's plaque was equivalent in amount to that of non-SF. In the cortex, glomerular obsolescence and interstitial fibrosis exceeded normal. Cystine crystallizes in BD with the probable result of cell injury, interstitial reaction, nephron obstruction, and with the potential of inducing cortical change and loss of IMCD tubular fluid pH regulation, resulting in apatite formation. The pattern of IMCD dilation, and loss of medullary structures is most compatible with such obstruction, either from BD lumen plugs or urinary tract obstruction from stones themselves.

Humans express three distinct collagenases, MMP-1, MMP-8, and MMP-13, that initiate degradation of fibrillar type I collagen. We have previously reported that ultraviolet irradiation causes increased expression of MMP-1, but not MMP-13, in keratinocytes and fibroblasts in human skin in vivo. We report here that ultraviolet irradiation increases expression of MMP-8 in human skin in vivo. Western analysis revealed that levels of the full-length, 85 kDa proenzyme form of MMP-8 increased significantly within 8 h post ultraviolet irradiation (2 minimal erythema doses). Increased full-length MMP-8 protein was associated with infiltration into the skin of neutrophils, which are the major cell type that expresses MMP-8. Immunofluorescence revealed coexpression of MMP-8 and neutrophil elastase, a marker for neutrophils. Immunohistology demonstrated MMP-8 expression in neutrophils in the papillary dermis between 4 and 8 h post ultraviolet irradiation, and in the epidermis at 24 h post radiation. MMP-8 mRNA expression was not detected in nonirradiated or ultraviolet-irradiated human skin, indicating that increased MMP-8 following ultraviolet irradiation resulted from preexisting MMP-8 protein in infiltrating neutrophils. Pretreatment of skin with the glucocorticoid clobetasol, but not all-trans retinoic acid, significantly blocked ultraviolet-induced increases in MMP-8 protein levels, and neutrophil infiltration. In contrast, all-trans retinoic acid and clobetasol were equally effective in blocking ultraviolet induction of MMP-1 and degradation of collagen in human skin in vivo. Taken together, these data demonstrate that ultraviolet irradiation increases MMP-8 protein, which exists predominantly in a latent form within neutrophils, in human skin in vivo. Although ultraviolet irradiation induces both MMP-1 and MMP-8, ultraviolet-induced collagen degradation is initiated primarily by MMP-1, with little, if any, contribution by MMP-8.

We have previously identified five Na, K-ATPase beta1-mRNA species that are expressed in the rat heart, kidney, and brain. These mRNAs which are unequal in their abundance have an identical coding region but differ in the length of their 5'- and 3'-untranslated regions (UTRs). In this study we examined the possibility that the beta1-mRNA species exhibit differential translational efficiencies. We constructed expression plasmids encoding each of the five mRNAs and transcribed and translated them in vitro. Using rabbit reticulocyte system we determined the translation of the different mRNAs under conditions optimized for each beta1-cRNA and under an equivalent (standard) condition. The longest beta1-cRNA species (initiating at the first transcription start site and ending at the last [fifth] poly(A) site) exhibited the lowest relative translational efficiency averaging 0.2 +/- 0.05 units/mol of cRNA compared to the shortest beta1-cRNA species initiating at the first transcription start site and ending at the first poly(A) signal (with an assigned relative value of 1.0). These results suggested that the different translation rates of beta1-mRNAs may be due to their 3'-UTRs. To further define the role of beta1-3'-UTR, chimeric luciferase constructs containing different segments of the beta1-3'-UTR were transiently transfected into Clone 9 cells. Compared to the chimeric construct containing the shortest beta1-3'-UTR segment (ending at the first poly(A) site), the construct containing the full-length beta1-3'-UTR exhibited a luciferase expression of 0.23 +/- 0.04. To control for potential changes in the abundance of the expressed chimeric mRNAs which may lead to differences in luciferase expression, luciferase activity was normalized against chimeric luciferase-mRNA content measured in mixtures of cells stably transfected with the above constructs. The ratio of luciferase activity/chimeric luciferase-mRNA content in cells expressing the construct containing the entire beta1-3'-UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containing beta1-3'-UTR up to the first poly(A) signal (P < 0.05). We conclude that the translational efficiency of the different beta1-mRNA species is negatively regulated by the 3'-UTR of the mRNA and that a regulating region appears to be localized between the second and fifth poly(A) signals of beta1-mRNA., (Copyright 2001 Academic Press.)

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

An increasing number of proteins are thought to self-splice post-translationally on the level of the polypeptide, producing two separate proteins from one gene, neither of which is the protein predicted from the gene sequence. The recent elucidation of the mechanism of splicing has led to the identification of a number of post-translational protein modifications that use similar chemical pathways.

Two new triterpenoid saponins named asterlingulatosides A and B were isolated from the whole plants of Aster lingulatus. Their structures were determined by spectroscopic data and chemical transformations to be 3-O-beta-D-glucopyranosyl-3 beta,16 alpha-dihydroxyolean-12-en-28-oic acid-28-O-alpha-L-O-arabinopyranoside and 3-O-beta-D-glucopyranosyl-3 beta,16 alpha-dihydroxyolean-12-en-28-oic acid-28-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside++ +. They showed inhibitory activity on DNA synthesis in human leukaemia HL-60 cells.

The pathogenesis of fibrosis in chronic renal allograft rejection remains unknown. Since TGF-beta 1 plays a key role in fibrogenesis, we studied a rat model of chronic allograft rejection that shows similarities to the structural lesion described in patients. We previously demonstrated an increased expression of TGF-beta 1 in human kidney biopsies with acute and chronic rejection. Recipients of renal allografts (F344-Lewis) and isografts (Lewis-Lewis) were sacrificed at 4, 8, 24 and 52 weeks. Characteristic histologic changes of chronic rejection developed in the allografts as early as four weeks and were accompanied by progressive albuminuria significant by eight weeks. Allografts showed a progressive increase in mRNA expression of TGF-beta 1 and matrix proteins during the 52 week course. Increased matrix deposition by immunofluorescence was mostly present in the interstitium and vessels early and in all kidney compartments later. The mRNA expression of plasminogen activator inhibitor, a protease inhibitor stimulated by TGF-beta 1, increased along with TGF-beta 1 and matrix proteins. These results suggest that the fibrosis of chronic renal allograft rejection is mediated, at least partly, by the dual action of TGF-beta 1 on matrix deposition and degradation.

A new triterpene saponin, shimadoside A, has been isolated from Kalimeris shimadae and its structure deduced as 3-O-beta-D-glucopyranosiduronic acid-3 beta, 16 alpha-dihydroxyolean-12-en-28-oic acid-28-O-beta-D-xylopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1--> 4)-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranoside by means of spectral data, especially NMR, including COSY, HMQC, HOHAHA and ROESY techniques, and chemical degradation.

A new phenolic glycoside, asterbatanoside A [p-hydroxyacetophenone-4-O-beta-D-xylopyranosyl-(1-->6)-beta-D- glucopyranoside], and two new triterpenoid saponins, asterbatanoside B [2 alpha,3 beta,23-trihydroxyolean-12-en-28-oic acid-28-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside] and asterbatanoside C [3-O-beta-D-glucopyranosyl-2 beta,3 beta,23-trihydroxyolean- 12-en-28-oic acid-28-O-beta-D-glucopyranoside] were isolated from the roots of Aster batangensis. Their structures were determined by spectroscopic methods and chemical evidence. The total synthesis of asterbatanoside A is also reported.

Three pentapeptides have been isolated from the roots of Aster tataricus and their structures elucidated on the basis of spectroscopic analysis as well as chemical and enzymatic methods.

Two new medicagenic acid saponins, named asterbatanoside J and K, were isolated from the roots of Aster batangensis. On the basis of chemical and spectral studies especially 2D NMR including COSY, HETCOR, HMQC, HOHAHA, TOCSY, ROESY and HMBC techniques, their structures were established as 3-O-beta-D-glucopyranosyl-(1--> 6)-beta-D-glucopyranosyl-2 beta, 3 beta-dihydroxy-olean-12-en-23 alpha, 28-dioic acid- 28-O-alpha-L-arabinopyranosyl-(1-->3)-alpha-L- rhamnopyranosyl-(1-->2)-beta-D-fucopyranoside and 3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl- medicagenic acid-28-O-beta-D-xylopyranosyl-(-->4)-[alpha-L- arabinopyranosyl-(1 -->3)-alpha-L-rhamnopyranosyl-(1-->2) -beta-D-fucopyranside, respectively.

Four new triterpene saponins, asteryunnanosides A, B, C and D, have been isolated from Aster yunnanensis and their structures deduced as 2 alpha,3 beta,23-trihydroxyolean-12-en-28-oic acid-28-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside, 2 alpha,3 beta,23-trihydroxyolean-12-en-28-oic acid-28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, 2 alpha,3 beta-dihydroxyolean-12-en-28-oic acid-28-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside and 2 alpha,3 beta-dihydroxyolean-12-en-28-oic acid-28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, respectively, by means of spectral and chemical data.

Four novel triterpenoid saponins named asterbatanoside F, G, H and I were isolated from the roots of Aster batangensis and their structures elucidated as 3-O-beta-D-glucopyranosyl-23-O-acetyl-bayogenin-28-O-beta-D-glu cop yranosyl- (1-->6)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-glucopyranoside, 3-O-beta-D-glucopyranosyl-bayogenin-28-O-beta-D-glucopyranosyl-(1- ->6)-[alpha-f1p4amnopyranosyl-(1-->2)]-beta-D-glucopyranoside, 3-O-beta-D-glucopyranosyl- (1-->3)-beta-D-glucopyranosyl-bayogenin-28-O-beta-D-glucopyranosyl -(1-->6)-beta-D-glucopyranoside, and 3-O-beta-D-glucopyranosyl-(1-->3)-beta-D- glucopyranosyl-23-O-acetyl-bayogenin-28-O-beta-D-glucopyranosyl-(1 -->6)-beta-D- glucopyranoside by means of spectral data, especially NMR including COSY, HETCOR, COLOC, HOHAHA, ROESY and selective INEPT techniques, and chemical evidence.

Calcium signals following activation of P2Y purinergic, alpha 1 adrenergic, and muscarinic cholinergic receptors were examined in individual astroglial cells. ATP, phenylephrine and carbachol, each increased intracellular calcium levels ([Ca2+]i) to similar amplitudes in the presence or absence of extracellular Ca2+. The dose-response relationship showed that less than an order of magnitude increase in ligand concentration led to maximal increase in [Ca2+]i from basal levels. Simultaneous application of multiple ligands did not produce additive effects on [Ca2+]i. These data suggested that different ligands released Ca2+ from common stores and that each of the ligands could cause maximal release. Application of a second ligand immediately after the first ligand produced an additional Ca2+ rise, suggesting that the Ca2+ stores were rapidly refilled and that receptor desensitization rather than Ca2+ depletion accounted for the rapid decline of the Ca2+ peak. Caged IP3 produced Ca2+ signals similar to those produced by ligands. For a given cell, both caged IP3 and ligands sometimes produced only one level of partial Ca2+ increases, suggesting the presence of a pool of high IP3-sensitive stores. Together, our results indicate that neuroligands tend to generate an all-or-nothing Ca2+ release from IP3 sensitive stores. The interactions between different receptor systems most likely occur at the level of IP3 accumulation.

A new oleanane-type triterpene saponin named asteryunnanoside E and a new acetylene glycoside named asteryunnanoside I have been isolated from the roots of Aster yunnanensis, and their structures elucidated as 3-O-beta-D-glucopyranosyl-bayogenin-28-O-beta-D- glucopyranosyl-(1-->2)-beta-D-glucopyranoside and 2Z,8E-decadiene-4,6-diyn-1-O-beta-D-glucopyranos yl-(1-->2)-beta-D- glucopyranoside by means of 1D and 2D NMR techniques (COSY, HETCOR, COLOC, HOHAHA and ROESY) and chemical transformations.

A new monoterpene glycoside, shionoside C, and a new oleanane-type triterpene glycoside, aster saponin G, were isolated from the roots of Aster tataricus. Their structures were elucidated on the basis of spectral and chemical methods. Shionoside C is L-endo-camphanol-8-3,5-isopropylidene-beta-D- apiofuranosyl-(1-6)-beta-D-glucopyranoside, aster saponin G is 3-O-[O-alpha-L-arabinopyranosyl-(1-6)-beta-D-glucopyranosyl]-2 beta, 3 beta, 16 alpha-trihydroxyolean-12-en-28-oic acid (asterogenic acid) 28-[O-beta-D-xylopyranosyl-(1-4)-alpha-L- rhamnopyranosyl-(1-2)-beta-D-xylopyranosyl]ester.

B72.3, a monoclonal antibody with reactivity against human adenocarcinomas was obtained from the Cytogen Corporation in the form of an immunoconjugate coupled with linker-chelator GYK-DTPA by using proprietary carbohydrate directed site specific chemistry. The immunoconjugate was radiolabeled with indium-111 or yttrium-90. A preclinical analysis was performed in 10 normal beagle dogs. The pharmacokinetics of intravenously administered indium- and yttrium-labeled immunoconjugates were compared serially in blood, bone marrow and urine samples. Compared to 90Y less of the 111In label ended up in urine and more was found in blood and bone marrow. Indium-labeled B72.3 GYK-DTPA had relatively higher uptake in most glandular tissues than 111In-labeled antiferritin immunoconjugate. Bone marrow toxicity was the dose limiting side effect after intravenous infusion of 90Y-labeled B72.3 GYK-DTPA. Toxicity was also observed in the liver but not in other organ systems. Recently other investigators obtained similar results with these immunoconjugates in human patients. A preclinical pharmacokinetic analysis of radioimmunoconjugates in beagle dogs provided useful information regarding bone marrow toxicity, liver toxicity and in vivo instability of the immunoconjugate. Data suggest that for future trials in human patients, a more stable chelated immunoconjugate for yttrium is needed to achieve less liver uptake and a better correlation with the 111In-labeled product than the 90Y-labeled B72.3 GYK-DTPA used in this investigation.

B72.3, a monoclonal antibody with reactivity against human adenocarcinomas, was coupled with linker-chelator GYK-DTPA using carbohydrate mediated conjugation chemistry and radiolabeled with yttrium-90. Single and double intravenous injections of radioimmunoconjugate were compared for acute and late normal tissue toxicity in 15 beagle dogs. The second injection was given 4 or 8 days after the first. Pharmacokinetics of the radioimmunoconjugate in blood, bone marrow and urine were similar for first and second injections. Only bone marrow (acute) and liver (late) toxicity were observed. Both liver and bone marrow toxicity were decreased by fractionation of the injections. After double injections, the total equitoxic dose was 15 and 60% higher for bone marrow and liver toxicity, respectively. The mechanisms of normal tissue protection offered by fractionated radioimmunoglobulin therapy (RIT) remain to be defined. Fractionated RIT will have a better therapeutic ratio than single injection RIT, if antitumor effects appear to be less susceptible to fractionation than normal tissues.