Arylsulfatase a activity and electrophoretic banding patterns from isolated human blood fractions

Arylsulfatase A (ASA) activity was studied from five blood fractions, leukocytes plus platelets (LKPL), leukocytes (LK), neutrophils (N), lymphocytes (LYM), and platelets (PL). No significant difference was found among the mean ASA specific activity values for the fractions. Electrophoretic examination revealed four distinct activity bands for the LKPL and PL fractions, while the LK, N, and LYM fractions showed only a single, broad peak, which indicates that the four-band pattern is associated with enzyme obtained from platelets. The PL fraction gave a clearer, more distinct banding pattern than the other four fractions. This clearly resolved electrophoretic banding pattern of ASA from pure platelet preparations was demonstrated for variant human ASA as well. These findings may be significant to future work investigating the biochemical genetics of ASA.     

Further studies on a human lung tumor-associated antigen. Comparison of antigens from different tumors.

A human lung tumor-associated antigen was purified to homogeneity from a crude cell-free extract of a human lung adenocarcinoma using standard biochemical procedures. In order to facilitate monitoring the recovery of antigen, trace amounts of previously purified and radioiodinated antigen from another lung tumor were added to the crude extract. The purified antigen was a glycoprotein and contained sialic acid. The antigen had a molecular weight of 76,000 and appeared to contain three subunits, each with a molecular weight of 25,000. The antigen had the following physical properties: Stokes radius, 39.4 A; S20,w, 4.24 S; D20,w, 5.15 x 10(-7) cm2 S-1; and a frictional ratio of 1.40. In addition, the purified, radioiodinated antigen retained complete immune reactivity since it could be quantitatively precipitated with specific immune serum. All of these properties were in close agreement with the properties of another antigen which was purified from a separate human lung tumor. Thus, it appeared from the biochemical and immunochemical criteria presented in this report that a common and identical antigen was isolated from two distinct human lung tumor extracts.     

Transferrin microheterogeneity as a probe in normal and disease states

Isoelectric focusing of iron saturated serum has been established as a convenient method for showing transferrin glycan microheterogeneity. In a clinical setting, the method is used in the detection of cerebrospinal fluid leakage, the screening for surreptitious alcohol abuse and in the diagnosis of the carbohydrate deficient glycoprotein syndrome. In normal physiological states it can also be used as a tool to probe for changes in N-glycosylation.     

Arylsulfatase A from human tissues contains an endo-beta-N-acetylglucosaminidase F-resistant oligosaccharide

Homogeneous arylsulfatase A from human placenta, liver and urine contains two nonidentical subunits of 59 and 54 kDa. The two subunits are immunologically identical. The relative amount of low molecular weight subunits is only 20-30% of the total enzyme protein. Treatment of the enzyme under various conditions with endo-beta-N-acetylglucosaminidase F results in a decrease in the apparent molecular weight of both subunits by 1-2 kDa. a value that corresponds to the loss of a single N-linked oligosaccharide. However, as judged by carbohydrate staining, endo-beta-N-acetylglucosaminidase F does not remove all carbohydrate from the subunits or from glycopeptides of arylsulfatase A. In contrast, human prostatic acid phosphatase, a glycoprotein with a high content of mannose, hybrid and complex oligosaccharides is completely deglycosylated under identical experimental conditions. Several attempts to deglycosylate arylsulfatase A by chemical methods were unsuccessful due to poor recovery of the protein. From the present studies we conclude that arylsulfatase A contains an endo-beta-N-acetylglucosaminidase F resistant, perhaps O-linked carbohydrate.     

Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.     

Chromatographically different type II collagens from human normal and osteoarthritic cartilage.

The genetic type of collagen was examined in both fresh and 2 to 4 month $\text{in vitro}$ cultured human normal and osteoarthritic articular cartilage. Disc electrophoresis indicated that all cartilage samples examined contained only type II collagen, however CM cellulose chromatography revealed chromatographic differences between the normal and pathological tissues.     

Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung.

The primary structures of human pulmonary surfactant-associated proteins SP-A, SP-B and SP-C isolated from lung lavage of patients with alveolar proteinosis exhibit significant differences from lung surfactant proteins isolated from lungs of healthy individuals. In contrast to SP-A from normal lungs, proteinosis SP-A was shown by SDS gel electrophoresis to contain large amounts of unreducibly cross-linked beta chains. Specific primary structure modifications of SP-C and SP-B proteins were established by direct molecular weight and structural analysis, using [252Cf]plasma desorption mass spectrometry (PD/MS) as the principal method. In comparison to normal lung surfactant SP-B, proteinosis SP-B showed a significantly increased molecular weight by approx. 500 Da for the unreduced protein dimer. SP-C proteins from normal lungs were identified to possess a bis-cysteinyl-5,6-(thioester)palmitoylated structure, and to contain a frayed N-terminus resulting in two sequences of 34 and 35 amino acid residues. In contrast, SP-C from proteinosis patients was modified by (i) partial or even complete removal of palmitate residues and (ii) additional N-terminal proteolytic degradation. These results indicate the presence of pathophysiological structure modifications, which are likely to occur in the alveolar space, and may lead to a reduced surfactant function.     

Purification of a mammary-derived growth factor from human milk and human mammary tumors

A growth factor, mammary-derived growth factor 1 (MDGF1), has been purified to apparent homogeneity from human milk. The factor is a pepsin-sensitive, reducing agent-insensitive protein with a molecular mass of 62 kDa and a pI of 4.8. An apparently identical factor has been isolated from human mammary tumors, suggesting that MDGF1 might be made by and act as an autocrine growth factor for mammary cells. High affinity receptors for MDGF1 have been detected on mouse mammary cells, normal rat kidney cells, and A431 epidermoid cells (KD = 2 X 10(-10) M). MDGF1 at picomolar levels stimulates the growth of mammary cells and greatly amplifies their production of collagen, apparently via elevating collagen mRNA levels, an effect that is demonstrated for normal rat kidney cells. The responsiveness of mammary cells to MDGF1 is attenuated when the cells are grown on a basement membrane collagen substratum, a component of the extracellular matrix upon which these cells normally rest in vivo. MDGF1 thus may regulate the production of new basement membrane as mammary epithelium invades the stroma during proliferation.     

Localization and activity of iNOS in normal human lung tissue and lung cancer tissue

Inducible nitric oxide synthase (iNOS) is one of three enzymes generating nitric oxide (NO) from the amino acid L-arginine. iNOS-derived NO plays an important role in several physiological and pathophysiological conditions. NO is a free radical which produces many reactive intermediates that account for its bioactivity. In the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Moreover, high concentrations of NO have been shown to increase nuclear factor kappaB (NF-kB) activation. Recent investigations of NO expression in tumor tissue indicated that, at least for certain tumors, NO may mediate one or more roles during the growth of human cancer. We have studied iNOS in two tissue groups: normal human lung tissue and human lung cancer tissue. We localized iNOS in these tissues by immunohistochemistry and tested the mRNA expression by RT-PCR, the protein level by Western blot, and the protein activity by radiometric analysis. The results demonstrate different expression, localization and activity of iNOS in normal versus tumor tissue. This is suggestive of a role for NO production from iNOS in human lung cancer because high concentrations of this short molecule may transform to highly reactive compounds such as peroxynitrite (ONOO-); moreover, through the upregulator NF-kB, they can induce a chronic inflammatory state representing an elevated risk for cell transformation to cancer.     

LINE-1 methylation patterns of different loci in normal and cancerous cells

This study evaluated methylation patterns of long interspersed nuclear element-1 (LINE-1) sequences from 17 loci in several cell types, including squamous cell cancer cell lines, normal oral epithelium (NOE), white blood cells and head and neck squamous cell cancers (HNSCC). Although sequences of each LINE-1 are homologous, LINE-1 methylation levels at each locus are different. Moreover, some loci demonstrate the different methylation levels between normal tissue types. Interestingly, in some chromosomal regions, wider ranges of LINE-1 methylation levels were observed. In cancerous cells, the methylation levels of most LINE-1 loci demonstrated a positive correlation with each other and with the genome-wide levels. Therefore, the loss of genome-wide methylation in cancerous cells occurs as a generalized process. However, different LINE-1 loci showed different incidences of HNSCC hypomethylation, which is a lower methylation level than NOE. Additionally, we report a closer direct association between two LINE-1s in different EPHA3 introns. Finally, hypermethylation of some LINE-1s can be found sporadically in cancer. In conclusion, even though the global hypomethylation process that occurs in cancerous cells can generally deplete LINE-1 methylation levels, LINE-1 methylation can be influenced differentially depending on where the particular sequences are located in the genome.     

RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects

In human cells, error-free repair of DNA double-strand breaks requires the DNA pairing and strand exchange activities of RAD51 recombinase. Activation of RAD51 recombination activities requires the assembly of RAD51 presynaptic filaments on the single-stranded DNA that forms at resected DSB ends. Mutations in proteins that control presynaptic filament assembly, such as BRCA2, and in RAD51 itself, are associated with human breast cancer. Here we describe the properties of two mutations in RAD51 protein that derive from human lung and kidney tumors, respectively. Sequence variants Q268P and Q272L both map to the DNA binding loop 2 (L2) region of RAD51, a motif that is involved in DNA binding and in the allosteric activation of ATP hydrolysis and DNA strand exchange activities. Both mutations alter the thermal stability, DNA binding, and ATPase properties of RAD51, however both variants retain intrinsic DNA strand exchange activity towards oligonucleotide substrates under optimized conditions. In contrast, both Q268P and Q272L variants exhibit drastically reduced DNA strand exchange activity in reaction mixtures containing long homologous ssDNA and dsDNA substrates and human RPA protein. Mixtures of wild-type and variant proteins also exhibit reduced DNA strand exchange activity, suggesting that heterozygous mutations could negatively affect DNA recombination and repair processes in vivo. Together, the findings of this study suggest that hypomorphic missense mutations in RAD51 protein could be drivers of genomic instability in cancer cells, and thereby contribute to the etiology of metastatic disease.     

Expression patterns of microRNAs 155 and 451 during normal human erythropoiesis

To clarify the roles of microRNAs (miRNAs) in erythropoiesis, the expression of miR-155, miR-221, miR-223, and miR-451 were analyzed during the differentiation of purified normal human erythroid progenitors in a liquid culture system. Cells increased almost 500-fold in a number, and differentiated to benzidine-positive mature erythroblasts. Analyses of miRNA expression using the quantitative real-time polymerase chain reaction showed that the expression level of miR-155 decreased about 200-fold, and that the expression of miR-451 increased about 270-fold during 12 days of cultures. A moderate down-regulation of miR-221 and miR-223 was observed. MiR-451 was expressed in red blood cells about 10⁴-fold more than in granulocytes, obtained from normal human peripheral blood. These observations suggest that miR-155 and miR-451 are key molecules for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be a relevant method for analyzing pathological erythropoiesis.