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Marine cloud brightening (MCB) is the deliberate injection of aerosol particles into shallow marine clouds to increase their reflection of solar radiation and reduce the amount of energy absorbed by the climate system. From the physical science perspective, the consensus of a broad international group of scientists is that the viability of MCB will ultimately depend on whether observations and models can robustly assess the scale-up of local-to-global brightening in today's climate and identify strategies that will ensure an equitable geographical distribution of the benefits and risks associated with projected regional changes in temperature and precipitation. To address the physical science knowledge gaps required to assess the societal implications of MCB, we propose a substantial and targeted program of research-field and laboratory experiments, monitoring, and numerical modeling across a range of scales.

The objective of this work was to develop a plastic film from food sources with excellent thermal, mechanical, and degradability performance. Corn starch (CS)/nata de coco (NDC) were hybridized with addition of glycerin as plasticizer at different weight ratio and weight percent, respectively. Sample analysis found that the hybridization of CS with NDC improved the film forming properties, mechanical and thermal, degradation properties, as well as hydrophobicity and solubility of the film up to 0.5:0.5 wt hybrid ratio. The properties of the films were highly affected by the homogeneity of the sample during hybridization, with high NDC amount (0.3:0.7 wt CS:NDC) showing poor hydrophobicity, and mechanical and thermal properties. The glycerin content, however, did not significantly affect the hydrophobicity, water solubility, and degradability properties of CS/NDC film. Hybridization of 0.5:0.5 wt CS/NDC with 2 phr glycerin provided the optimum Young's modulus (15.67 MPa) and tensile strength (1.67 MPa) properties., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: The level of food specific IgG antibody in serum of patients with suspected food intolerance in Beijing area was detected and analyzed, so as to provide a basis for patients to eliminate the food that causes disease in their diet. Methods: Data were retrospectively analyzed in a total of 2368 suspected chronic food intolerance patients (all resident population in Beijing) admitted to Beijing Jishuitan Hospital from October 2015 to December 2019, including 859 males and 1509 females. The age range ranged from 2 months to 90 years, (40.2±18.6) years, and the median age was 36 years. Serum specific IgG (sIgG) antibody concentrations in 14 kinds of food (beef, chicken, pork, cod, shrimp, crab, egg, milk, corn, wheat, mushroom, soybean, tomato and rice) were determined by enzym-linked immunosorbentassay (ELISA).SPSS V22.0 was used for statistical analysis, and χ 2 was used to compare the positive rate of each group. Results: The positive rate of food sIgG in 2 368 patients was 82.7% (1959/2368), the higher sIgG positive rates were found in eggs (54.7%), shrimp (29.0%) and milk (26.0%), and the lowest sIgG positive rates were found in pork (4.1%) and beef (1.2%) ( P <0.05).There was significant gender difference in egg (χ²=10.885) and shrimp (χ²=6.607) sIgG, and female was significantly higher than male ( P <0.05).The sIgG positive rates of egg (χ²=515.645), shrimp (χ²=74.20.4), milk (χ²=305.65), soybean (χ²=39.413), wheat (χ²=133.222), cod (χ²=25.540), crab (χ²=55.521), corn (χ²=20.645), tomato (χ²=118.108), chicken (χ²=32.821), pork (χ²=26.539), beef (χ²=21.632) and rice (χ²=25.335) were significant in different age groups. The positive rate of shrimp and crab sIgG increased with age ( P <0.05).The positive rate of tomato sIgG decreased with age, and then increased slightly after 45 years. However, egg, milk, soybean, wheat, corn, and rice all showed a decreasing positive rate with the increase of age. The increase of chicken, pork and cod was observed in childhood, and then decreased with age, and then increased in middle and old age. Conclusions: There may be a certain correlation between food intolerance and elevated food sIgG levels in Beijing area. It is suggested that patients of different genders and ages should be optimized and adjusted according to their own characteristics, and children patients are still the key population for food intolerance control.

Even though desert dust is the most abundant aerosol by mass in Earth's atmosphere, atmospheric models struggle to accurately represent its spatial and temporal distribution. These model errors are partially caused by fundamental difficulties in simulating dust emission in coarse-resolution models and in accurately representing dust microphysical properties. Here we mitigate these problems by developing a new methodology that yields an improved representation of the global dust cycle. We present an analytical framework that uses inverse modeling to integrate an ensemble of global model simulations with observational constraints on the dust size distribution, extinction efficiency, and regional dust aerosol optical depth. We then compare the inverse model results against independent measurements of dust surface concentration and deposition flux and find that errors are reduced by approximately a factor of two relative to current model simulations of the Northern Hemisphere dust cycle. The inverse model results show smaller improvements in the less dusty Southern Hemisphere, most likely because both the model simulations and the observational constraints used in the inverse model are less accurate. On a global basis, we find that the emission flux of dust with geometric diameter up to 20 μm (PM 20 ) is approximately 5,000 Tg/year, which is greater than most models account for. This larger PM 20 dust flux is needed to match observational constraints showing a large atmospheric loading of coarse dust. We obtain gridded data sets of dust emission, vertically integrated loading, dust aerosol optical depth, (surface) concentration, and wet and dry deposition fluxes that are resolved by season and particle size. As our results indicate that this data set is more accurate than current model simulations and the MERRA-2 dust reanalysis product, it can be used to improve quantifications of dust impacts on the Earth system., Competing Interests: Competing interests The authors declare that they have no conflict of interest.

Circular RNAs (circRNAs) exert pivotal effects on regulating the progression of osteosarcoma (OS). It was found through microarray analysis that circ-0002052 is abnormally expressed in OS, but the role of circ-0002052 in OS remains obscure. The results of this research manifested that relative to that in non-tumor controls, circ-0002052 level was raised in OS tissues. Up-regulated circ-0002052 was associated with advanced stage, tumor size, and metastasis. Additionally, circ-0002052 elevation indicated a low survival rate in OS patients and silencing of circ-0002052 suppressed proliferation, migration, and invasion of OS cells. It was proved that circ-0002052 sponged miR-382 and stimulated STX6 expression, thus activating Wnt/β-catenin. The function of circ-0002052 reduction in OS cells was effectively reversed by miR-382 suppression. To sum up, it can be concluded that circ-0002052, functioning as a sponge for miR-382, enhances the activation of Wnt/β-catenin mediated by STX6 to stimulate the progression of OS, and circ-0002052 may be an underlying treatment target and a biomarker for prognosis of osteosarcoma.

Invasive plants are a huge burden on the environment, and modify local ecosystems by affecting the indigenous biodiversity. Invasive plants are generally less affected by pathogens, although the underlying molecular mechanisms responsible for their enhanced resistance are unknown. We investigated expression profiles of three defense hormones (salicylic acid, jasmonic acid, and ethylene) and their associated genes in the invasive weed, Alternanthera philoxeroides , and its native congener, A . sessilis , after inoculation with Rhizoctonia solani . Pathogenicity tests showed significantly slower disease progression in A . philoxeroides compared to A. sessilis . Expression analyses revealed jasmonic acid (JA) and ethylene (ET) expressions were differentially regulated between A . philoxeroides and A. sessilis , with the former having prominent antagonistic cross-talk between salicylic acid (SA) and JA, and the latter showing weak or no cross-talk during disease development. We also found that JA levels decreased and SA levels increased during disease development in A . philoxeroides . Variations in hormonal gene expression between the invasive and native species (including interspecific differences in the strength of antagonistic cross-talk) were identified during R. solani pathogenesis. Thus, plant hormones and their cross-talk signaling may improve the resistance of invasive A . philoxeroides to pathogens, which has implications for other invasive species during the invasion process.

Bone marrow mesenchymal stem cells (BMSCs) are considered the most important seed cells in bone tissue engineering. The present study aimed to investigate the potential of rabbit BMSCs in osteogenesis after the transfection of human BMP-2 and EGFP recombinant adenovirus. Rabbit BMSCs were isolated and the surface stem cell makers, including CD29, CD44 and CD45 were detected by flow cytometry. The expression of BMP-2 mRNA and protein in BMSCs were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. After an induction with osteogenic medium, the alkaline phosphatase (ALK) activity at 7 days, the type I collagen at 14 days, and the calcium nodules at 21 days were performed using an ALK activity kit, immunohistochemical staining and alizarin red S staining, respectively. The expression levels of proteins related to the Wnt signaling pathway were detected by western blot analysis. The positive rates of CD29, CD44 and CD45 were 62.92±1.99, 93.55±0.99 and 0.21±0.12%. The expression of BMP-2 mRNA and protein was significantly upregulated in Ad-BMP-2/EGFP transfected BMSCs. Furthermore, Ad-BMP-2/EGFP induced ALP activity, promoted the production of type I collagen and calcium nodule formation in rabbit BMSCs. The levels of β-catenin, cyclin D1, Runx2 and c-myc were upregulated in Ad-hBMP-2/EGFP transfected BMSCs, while the level of GSK3β was significantly decreased. Results also indicated that the overexpression of BMP-2 by Ad-hBMP-2/EGFP enhanced the osteogenic differentiation ability of cultured rabbit BMSCs via stimulating the Wnt signaling pathway with the accumulation of β-catenin and suppression of GSK3β. The Ad-hBMP-2/EGFP transfected rabbit BMSCs are expected to be a good seed cell in bone tissue engineering.

The Kunming dog is the first and only working dog breed from China to be recognized worldwide. As a domestic working dog, its excellent working performance has been well established; however, its normal reproductive parameters are not well understood. Therefore, this study was conducted to document the main reproductive parameters of this purebred working dog in field breeding conditions. Data on 1004 heats (753 with mating) from 203 bitches between 2008 to 2014, were collected and analyzed. The pregnancy rate and whelping rate was 79.42% and 75.30%, respectively. Finally, for 567 litters (4298 puppies), the mean litter size was 7.19 ± 0.12 puppies (range 1-15). The mean gestation period and birth weight were approximately 61.64 ± 0.10 days and 407.25 ± 1.21 g. The mean sex ratio was 1.03 males to 1.00 female. Estrus occurred throughout the year with no significant differences between seasons and months (P > 0.05), which confirms that Kunming dogs are non-seasonal breeders; births occurred in every month of the year. Pregnant bitches exhibited significantly longer inter-estrus intervals than non-pregnant bitches (220.85 ± 2.05 vs. 180.19 ± 2.94 days, P < 0.05). Bitch parity influenced litter size, and the gestation length and birth weight of the puppies were negatively affected by litter size. This study helps elucidate the reproductive potential of this breed and provides reference values for reproductive performance in the Kunming dog., (© 2017 Japanese Society of Animal Science.)

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen-thawed spermatozoa of 100 × 10 6 unsorted (a dose in practice) and 4 × 10 6 sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm-sexing technology potentially applicable for elite breeding units., (© 2017 Japanese Society of Animal Science.)

Objective: To investigate the regulation of JAK2 tyrosine kinase inhibitor ruxolitinib on extracellular matrix metalloproteinase (MMP in JAK2V617F positive myeloproliferative neoplasms (MPN) cells. Methods: ①Forty cases of newly diagnosed JAK2V617F positive MPN patients and 15 healthy volunteers as control in Baoding No.1 Hospital between January 2012 and December 2015 were enrolled in this study. JAK2V617F/JAK2 ratio was detected by real-time-PCR; the expression levels of phosphorylation protein tyrosine kinase 2 (p-JAK2) , MMP-2 and MMP-9 in pathological tissues of bone marrow were detected by immunohistochemistry. The bone marrow cells of JAK2V617F positive MPN patients were treated with ruxolitinib, then the migration ability and MMP-2, MMP-9 gene and protein expression levels were detected. ②The human erythroleukemia cell line HEL cells were treated with different concentrations of ruxolitinib (0, 50, 100, 250, 500, 1 000 nmol/L) . The cell viability was detected by CCK-8 test; cell migration ability was tested by transwell chambers. The mRNA expression levels of JAK2, MMP-2 and MMP-9 were detected by real-time-PCR. The protein expression levels of p-JAK2, MMP-2 and MMP-9 were detected by Western blot. Results: ①The expression levels of p-JAK2, MMP-2 and MMP-9 in the newly diagnosed group were significantly higher than control group respectively [ (78.56±24.55) % vs (41.59±17.29) %, P <0.05; (48.25±18.74) % vs (22.79±13.89) %, P <0.05; (53.29±19.28) % vs (15.56±14.96) %, P <0.05]. Spearman correlation analysis showed the positive correlation of MMP-2 and MMP-9 protein expression levels with JAK2V617F mutation ( r =0.526, P =0.001; r =0.543, P =0.001) . ② The proliferation of HEL cells was inhibited by different concentrations of ruxolitinib in time and dose dependent manner. ③Cell migration test showed the number of cells leaked to the low chamber in MPN patients bone marrow cells and HEL cells treated with 5 nmol/L of ruxolitinib group were significantly lower than that without ruxolitinib treatment after 24 h [ (154.7±27.5) vs (320.3±67.3) , t =13.47, P <0.05; (70.7±10.5) vs (135.3±16.7) , t =13.89, P <0.05]. The mRNA and protein expression levels of JAK2, MMP-2 and MMP-9 decreased with the increased concentration of ruxolitinib. Conclusion: Ruxolitinib inhibits MPN cell migration and expression of MMP-2 and MMP-9 via JAK2 signal pathway.

Aim: To investigate the effect of tyrosine kinase inhibitor imatinib mesylate on the PTEN signaling pathway and the cell invasion in K562 cells., Methods: K562 cells were treated with different concentrations of imatinib mesylate. After different time periods, the mRNA levels of BCR/ABL, PTEN and FAK were detected by real-time fluorescent quantitative PCR (FQ-PCR) to analyze their relationships. The protein level of FAK was detected by immunocytochemistry. The cell invasive ability was examined by Transwell (Boyden chamber) assay., Results: In the initial 36 h, the expression level of PTEN mRNA was up-regulated and the FAK mRNA was down-regulated with the reduction of BCR/ABL fusion gene expression and the cell invasive ability of K562 cells was inhibited by 2 μg/mL imatinib mesylate. 48 h later, the PTEN mRNA expression level decreased and the FAK mRNA expression level was elevated with the restore of BCR/ABL fusion gene. BCR/ABL mRNA level presented a positive correlation with PTEN mRNA expression level, and a negative correlation with FAK mRNA., Conclusion: Tyrosine kinase inhibitor imatinib mesylate can regulate PTEN/FAK pathway and inhibit the leukemia K562 cell invasive ability via restraining BCR/ABL fusion gene.

Objective: To explore the effects of tumor-suppressing gene wild type PTEN on the cell proliferation, apoptosis and the possible regulations of apoptosis-related molecules Survivin, Xiap and Smac gene in human chronic myeloid leukemia (CML) and cell line K562 cells., Methods: (1) The recombinant adenovirus containing green fluorescent protein (GFP) and PTEN (Ad-PTEN-GFP) or empty vector (Ad-GFP) was transfected into K562 cells. The growth of K562 cells was observed by MTT assay while cell cycle and apoptotic rate were assessed by flow cytometry (FCM). PTEN, Survivin, Xiap and Smac mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR) while PTEN protein levels analyzed by Western blot. (2) The expression levels of PTEN, Survivin, Xiap and Smac mRNA were detected in 10 chronic myelogenous leukemia (CML) patients in chronic phase (CML-CP), 10 CML patients in blast crises (CML-BC) and 10 normal control marrow mononuclear cells (MMNC)., Results: The growth of K562 cells was suppressed markedly. And the maximal growth inhibition rate was 38.6% after the transfection of PTEN. Survivin, Xiap, Smac mRNA expression levels were down-regulated by around 6.14, 7.44 and 2.95 folds respectively (0.0700 ± 0.0059, 0.0089 ± 0.0006, 0.0600 ± 0.0039 vs 0.4370 ± 0.0790, 0.0661 ± 0.0072, 0.1580 ± 0.0078 vs 0.4530 ± 0.0810, 0.0700 ± 0.0079, 0.1770 ± 0.0085, all P < 0.01). The mRNA expression level of PTEN in CML-BC patients was lower than that in CML-CP patients and normal control. But Survivin, Xiap, Smac mRNA expression levels were higher in CML-BC patients than those in CML-CP and normal control., Conclusion: The over-expression of PTEN gene may inhibit the proliferation of K562 cells and promote cell apoptosis via the regulation of Survivin, Xiap and Smac genes.

Comprehensive and structured annotations for all genes on a microarray chip are essential for the interpretation of its expression data. Currently, most chip gene annotations are one-line free text descriptions that are often partial, outdated and unsuitable for large-scale data analysis. Therefore the interpretation of microarray gene expression clusters is often limited. Although researchers can manually navigate a collection of databases for better annotations, it is only practical for limited number of genes. Existing meta-databases fail to provide comprehensive categorized annotations for hundreds of genes simultaneously. We have developed an automatic system to address this issue. GeneView system monitors various data sources, extracts gene information from a source whenever it is updated, comprehensively matches genes, and integrates them into a central database by categories, such as pathway, genetic mapping, phenotype, expression profile, domain structure, protein interaction, disease association, and references. The system consists of four major components: (1) relational database; (2) data processing; (3) user curation; (4) data query. We evaluated it by analyzing genes on cDNA and Affymetrix Oligo chips. In both cases, the system provided more accurate and comprehensive information than those provided by the vendors or the chip users, and helped identify new common functions among genes in the same expression clusters.

Background: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of ganglion cells in the nerve plexuses of the lower digestive tract. HSCR has a complex pattern of inheritance and is sometimes associated with mutations in genes of the receptor tyrosine kinase (RET) and endothelin receptor B (EDNRB) signaling pathways, which are crucial for development of the enteric nervous system., Methods: Using PCR amplification and direct sequencing, we screened for mutations and polymorphisms in the coding regions and intron/exon boundaries of the RET, GDNF, EDNRB, and EDN3 genes of 84 HSCR patients and 96 ethnically matched controls., Results: We identified 10 novel and 2 previously described mutations in RET, and 4 and 2 novel mutations in EDNRB and in EDN3, respectively. Potential disease-causing mutations were detected in 24% of the patients. The overall mutation rate was 41% in females and 19% in males (P = 0.06). RET mutations occurred in 19% of the patients. R114H in RET was the most prevalent mutation, representing 7% of the patients or 37% of the patients with RET mutations. To date, such a high frequency of a single mutation has never been reported in unrelated HSCR patients. Mutations in EDNRB, EDN3, and GDNF were found in four, two, and none of the patients, respectively. Two patients with mutations in genes of the EDNRB pathway also harbored a mutation in RET. Three novel and three reported polymorphisms were found in EDNRB, EDN3, and GDNF., Conclusion: This study identifies additional HSCR disease-causing mutations, some peculiar to the Chinese population, and represents the first comprehensive genetic analysis of sporadic HSCR disease in Chinese.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

The major obstacle of differential display is not the technique itself but rather the post-differential display issueof discriminating between false positives and the truly differentially expressed mRNAs. This process is arduous and requires large amounts of RNA. We present and validate a method which allows one to screen putative positives from differential display analysis using only micrograms of total RNA. More importantly, we demonstrate that cDNA probes generated from amplified RNA are representative of the starting mRNA population and can be used for differential screening of mRNA species at a detectable limit of sensitivity of>/=1/40 000.

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

Horseradish peroxidase (HRP) was conjugated to nondegradable polycationic poly(D-lysine) (PDL) through either a thioether (HRP-S-PDL) or a disulfide (HRP-SS-PDL) linkage. The binding and transcytosis of these conjugates was studied in Madin-Darby canine kidney (MDCK) cell monolayers grown on 3-microns microporous polycarbonate filters. Conjugation of HRP to PDL with both linkages markedly increased the binding of this protein onto the cell monolayers. However, an enhancement of the transcellular transport of HRP in both apical-to-basal and basal-to-apical directions was observed only in HRP-SS-PDL, but not in HRP-S-PDL. HRP-SS-PDL transport was inhibited by colchicine and by 4 degrees C incubation. The transport of 14C-sucrose was not affected by the presence of conjugates. These results indicate that the transport of the conjugate across the cell monolayers was due to a transcellular process rather than to any leakage of the cell junction caused by polycations. The disulfide linkage between HRP and PDL was cleaved rapidly at the basal and, to a lesser extent, at the apical surface of the cell. Neuraminidase treatment decreased the binding of the conjugates onto the cell surface, but did not decrease the transcellular transport, suggesting that not all surface-bound conjugates were available for transcytosis. These results demonstrate that disulfide linkages can be cleaved during transcytosis in MDCK cells. The cleavage, however, occurs mostly at the binding site on the cell surface, which may prevent the cellular uptake of the intact conjugate.

The transcytosis of horseradish peroxidase, as well as its poly(L-lys) and poly(D-lys) thioether conjugates, was investigated in Strain I Madin-Darby canine kidney (MDCK) cell monolayers grown on 0.4 microns pore size polycarbonate membranes in Costar Transwells. The 3 types of HRP had almost identical rates of transport during the first 2 hr of incubation. However, a significant increase of basal-to-apical transport was detected beginning at 3 hr only in Transwells containing the poly(L-lys) conjugate. This increase was inhibited by colchicine (2 microM) and by the Bowman-Birk protease inhibitor (0.1 mg/ml), but not by NH4Cl (10 mM) or chloroquine (0.1 mM). The increase was abolished either by prior trypsinization of the conjugate or by incubation at 4 degrees C. Ultrafiltration studies indicated that the transcytosed poly(L-lys) conjugate was smaller in size than the original conjugate. These results indicate that the conjugate was processed during transcytosis in a non-lysosomal proteolytic compartment, where its poly(L-lys) moiety was selectively degraded, allowing active peroxidase to be released into the apical medium.