Biomedical subjects
Publications and source records attributed to M Garcia.
An adhesin with fimbria-like properties was present in four strains of verocytotoxigenic Escherichia coli isolated from diarrhoeic calves in Brazil.
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In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75-1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin-D-like aspartyl protease bearing mannose-6-phosphate signals. This precursor displays an in vitro autocrine mitogenic activity on estrogen-deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin-D-like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin-B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin-L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.
The development of the first meiotic prophase stages was studied in two series of human female embryos and fetuses aborted for social reasons. The first series (64 embryos or fetuses aborted at 6-24 weeks of gestation) was used mainly to perfect the methods applied to obtain chromosome preparations and synaptonemal complex spreads. The second series (37 embryos or fetuses aborted at 9-24 weeks of gestation) was used to establish the timing and to characterize the different stages of prophase I. Leptotene-zygotene figures were observed in some embryos at 10 weeks of gestation. Typical zygotene figures were seen at 11-22 weeks. Pachytenes were first observed at 12-13 weeks, and the proportion of these figures was usually lower than 40%. Diplotenes were seen in fetuses with a gestational age of 14 weeks or more. The duration of the process in the human female is thus about 3-4 weeks, a similar period to that described for the male.
We have previously described a secreted glycoprotein of mol. wt 52,000 (52-kDa protein) which is induced by estrogen in some human breast cancer cell lines. This protein has been identified as the proenzyme of a lysosomal cathepsin-D-like protease which is secreted in large proportions in breast cancer cells. To determine which information may be generated by this marker when detected in mammary tumors, in comparison with hormone receptors, we used monoclonal antibodies interacting specifically with the 52-kDa protein and its related cellular processed products (mols. wts 48 and 34 kDa). A high concentration of this protein has been shown in proliferative ductal mastopathies and cysts, suggesting its value in detecting high-risk mastopathies. We now present the immunoperoxidase distribution of this protein in breast carcinoma compared to the cytosolic hormone receptors assayed in parallel. In 232 breast cancers, no correlation was found between the cellular 52-kDa protein content and cytosolic estrogen or progesterone receptor concentrations. This absence of correlation was also shown by the constitutive production of this protein by estrogen-receptor-negative breast cancer cell lines and confirmed by double immunostaining of breast cancer cell aspirates showing a dissociation between the cytoplasmic staining of this 52-kDa lysosomal protease and the nuclear staining of the estrogen receptor. These clinical results, associated with the in vitro mitogenic and proteolytic activities of this protein, strongly suggest that the 52-kDa protein staining in tissue is associated with tumor proliferation and/or invasion, rather than with hormone responsiveness.
A retrospective study was done to determine if a reduction in daily grain consumption or decline in daily milk yield were accurate indicators of estrus in 25 Holstein cows. A computer automatically identified all cows that left more than 10% of their daily grain allocation in the feed stall or that gave less than 85% of their most current 7-d daily average milk yeild. By retrospectively analyzing breeding records, grain refusal and milk declines were identified as either occurring in conjunction with estrus or nonestrus. Declines in grain consumption and milk yield were frequently associated with estrus, but many cows in estrus did not exhibit such declines. While such declines hold promise as estrous detection aids, it was concluded that refusals of daily grain allocation or declines in daily milk yield were not sufficiently accurate to supplant other methods of estrous detection in dairy cattle.
We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis. In estrogen receptor positive human breast cancer cell lines (MCF7, ZR75-1) estrogens specifically increase the production into the culture medium of a 52,000 daltons (52K) glycoprotein. Several high affinity monoclonal antibodies to the partially purified secretory 52K protein have allowed to purify to homogeneity this protein and its cellular processed products. The 52K protein has been identified as the secreted precursor of a cathepsin-D like protease bearing mannose-6-phosphate signals and routed to lysosomes via mannose-6-phosphate receptor. The protease is mitogenic in vitro on estrogen deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its activation. The cellular related proteins, as detected by immunohistochemistry and immunoassay are more concentrated in proliferative mammary ducts than in resting ducts and their concentration in breast cancer cytosol appears to be more correlated with lymph nodes invasion and disease free survival (with S. Thorpe, Copenhagen) than with the estrogen receptor (RE) level. The protein is also produced constitutively by RE-negative cell lines, while in some antiestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in lambda gt11 has allowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the normal human kidney cathepsin-D.(ABSTRACT TRUNCATED AT 250 WORDS)
It is well-known that uremic patients present prolonged bleeding times as a common complication. Factors responsible for this disorder have been extensively investigated. In order to elucidate the possible role of uremic middle molecules as responsible for the bleeding tendency observed in uremia, we have studied the effect on platelet aggregation of middle molecules obtained from uremic plasma by Sephadex and ion-exchange chromatography. Our results show that some purified middle molecular fractions have a specific inhibitory activity on platelet aggregation and suggest an important role for these compounds in the pathogenesis of uremic bleeding.
The double immunohistochemical detection of estrogen receptor (ER) and the estrogen-regulated 52,000-dalton protein (52kDa-P) was performed on 35 fine needle aspirates of primary breast cancers. The nuclear ER, stained brown, was differentiated from the 52kDa-P, stained in red, in the cytoplasm of the same cells. The significant correlation obtained between the percentages of positive aspirated cells and the cytosolic concentrations of these two markers showed the validity of semiquantitative detection in fine needle aspirates, which appeared to be more representative of the content of these markers in tumors than was the detection in a single tissue section. Tumor heterogeneity also was defined by this double immunostaining, since among the tumors positive for both ER and 52kDa-P (18 of 35 cases), a heterogeneous group of 12 tumors contained the markers in two distinct cell populations, whereas a more homogeneous group of 6 tumors contained double-stained cells. The first information provided by this study is that the 52kDa-P is not correlated with the ER. This absence of correlation is in accordance with other studies and indicates that the 52kDa-P is associated with tumor proliferation and possibly invasion, rather than with hormone responsiveness or differentiation. The feasibility and validity of double immunostaining of these two markers in human breast cancer aspirates should stimulate larger studies--with clinical follow-up of the patients--aimed at establishing the clinical usefulness of this presurgical test.
Basic studies developed from human cell lines or tissue biopsies have allowed to isolate monoclonal antibodies and complementary DNAs specific for tumoral antigens and rare proteins implicated in the control of cancer growth and invasiveness. Present clinical studies indicate that some molecular probes display a potential value as markers of tissue specificity, of hormone dependency or prognosis in breast and endometrial cancers.
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Erythrocyte pyruvate kinase deficiency was detected in a Cuban girl with congenital nonspherocytic haemolytic anemia. Kinetic and immunochemical studies showed that the case was different from those hitherto reported. The variant(s) was tentatively designated PK "Alquizar", following the recommendations of the International Committee for Standardization in Hematology.
An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.
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The BALB/cf/Cd substrain of mice, developed by inbreeding and selection, shows a 70% incidence of spontaneous kidney adenocarcinomas. Initially foster-nursed on C3H mothers, these mice no longer produce mammary tumors, but there is evidence that mouse mammary tumor virus (MMTV) is involved in the formation of these renal carcinomas. We identified a chromosomal region called int-41, representing a locus interrupted by the integration of an exogenous MMTV provirus in a BALB/c mammary tumor. We found a DNA rearrangement and transcriptional activation in the domain specified by the int-41 probe in one primary kidney adenocarcinoma. A 5.2-kb int-41 specific mRNA was detected in the kidney tumor cell line established from a transplantable renal adenocarcinoma. This mRNA hybridized with MMTV and int-41 specific probes suggesting that it is a hybrid molecule composed of MMTV-LTR sequences covalently linked to host cell RNA. This mRNA was strongly stimulated by the presence of glucocorticoid hormone in the culture medium. Our data are compatible with the hypothesis that the int-41 chromosomal domain is involved in the neoplastic transformation of epithelial cells from different organs.
Three experiments were conducted in order to investigate the role of brain androgen and estrogen receptors in sex hormone activated male reproductive behavior in Japanese quail. In Experiment 1, castrated male quail were injected with oil, testosterone propionate (TP), flutamide (FLUT), an androgen antagonist, or TP+FLUT. Males given TP+FLUT, compared with birds receiving TP alone, strutted much less and had smaller proctodeal (foam) glands. Copulation was reduced by FLUT only on the last test day and only on one measure (number of head grabs + mounts). These results suggest that binding of testosterone or one of its metabolites to an androgen receptor is part of the mechanism of TP activated strutting, and therefore that central nervous system androgen receptors are involved in a male reproductive behavior pattern. In Experiment 2, castrated male quail were injected with oil, with 50 micrograms estradiol benzoate (EB), or with 25, 50 or 100 micrograms diethylstilbestrol (DES), a synthetic estrogen that does not bind to androgen receptors. EB but not DES activated copulation to a significant extent. In Experiment 3 male and female quail with photically regressed gonads were given intraperitoneal Silastic implants of DES, estradiol (E) or cholesterol. DES was highly effective at activating male-typical copulation in males and receptivity in both sexes. Thus hormonal interaction with estrogen receptors alone is sufficient for the activation of male-typical as well as female-typical copulatory behavior in this species.
A partial hepatectomy (an average of 36% of hepatic mass removed) was performed in rainbow trout. Thirty days after this partial hepatic removal, the liver had recovered its initial weight. During regeneration the remaining liver was unable to maintain normal blood levels of protein, cholesterol and, partially, lipids which decrease after surgery. The results obtained show that functional and liver weight regeneration proceed at different rates throughout a given time course, weight recovering faster than complete functional restoration.
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