Comparative analysis of biotin intranuclear inclusions of gestational endometrium using the APAAP, ABC and the PAP immunodetection systems.

AIMS/BACKGROUND: Intranuclear inclusions have been observed in gestational endometrial glands. Although resembling Herpes simplex virus infected cells, these nuclei have been shown to contain endogenous biotin. Hence, immunohistochemical systems using the avidin-biotin complex will result in cross reaction and false positivity. This study investigates a series of 10 gestational endometria (formalin fixed and paraffin wax embedded) with intranuclear inclusions using three different immunodetection systems with primary anti-biotin. RESULTS: Both the alkaline phosphatase anti-alkaline phosphatase (APAAP) and peroxidase anti-peroxidase (PAP) systems confirmed that the intranuclear inclusions were endogenous biotin. The streptavidin biotin complex (StreptABC) system produced a positive reaction in both test sections and controls (omitting primary anti-biotin) in the presence of prior blocking with free avidin and biotin. In addition, aberrant immunoreactivity was observed in adjacent nuclei/cytoplasm of endometrial glands and decidualised stroma in the negative control of the PAP system. This was subsequently eliminated using microwave pretreatment prior to immunohistochemistry. CONCLUSION: The use of either the APAAP or PAP immunodetection systems (the latter with microwave pretreatment) is recommended for any immunohistochemical or non-isotopic in situ hybridisation investigation undertaken on gestational endometria.     

Argyrophil cells in normal, hyperplastic, and neoplastic endometrium.

Scanty argyrophil cells are present in a substantial proportion of normal endometria, particularly during the secretory stage of the cycle. Argyrophil cells are also present in the various types of hyperplastic endometria and are found in more than half of endometrial adenocarcinomas. In some endometrial neoplasms they are present in abundance, but tumours rich in such cells do not have any features suggestive of a carcinoid tumour and are morphologically identical to adenocarcinomas of similar grade which are devoid of argyrophil cells. Endometrial adenocarcinomas containing argyrophil cells tend to be well differentiated and tend not to invade deeply into the myometrium. It is suggested that Müllerian epithelial stem cells possess a potentiality for differentiation into APUD cells.     

Application of immunohistochemistry in the evaluation of neoplastic epithelial lesions of the uterine cervix and endometrium

Routine use of immunohistochemistry has contributed greatly to the evaluation of neoplastic epithelial lesions of the uterine cervix and endometrium. This review highlights recognized diagnostic applications of these markers, addresses certain diagnostic pitfalls, and documents recent advances in the use of immunohistochemistry in the evaluation of cervical squamous intra-epithelial lesions (SILs), endocervical glandular neoplasia, basaloid cervical tumours, endometrial adenocarcinomas and their differential diagnoses. Increased MIB-1 staining is useful in distinguishing SILs and endocervical neoplasia (in situ or invasive) from benign mimics, such as cervical atrophy and tubal metaplasia, respectively. Immunohistochemisty with antibodies to vimentin, carcino-embryonic antigen (CEA) and oestrogen receptor (OR) may discriminate between invasive endocervical adenocarcinoma (ECA) and endometrial adenocarcinoma (EMC) in limited curettage specimens. ECAs tend to exhibit a vimentin-negative, CEA-positive, OR-negative profile, while the opposite is true for EMCs. Staining for MIB-1, OR and p53 may offer additional prognostic information in EMCs.     

Differential expression and localization of de-novo synthesized endometriotic haptoglobin in endometrium and endometriotic lesions

Endometriosis protein-I (ENDO-I) mRNA expression and protein localization were evaluated using in-situ hybridization and immunohistochemistry in endometriotic lesions and eutopic endometrium from women with endometriosis, and in eutopic endometrium from women without endometriosis (controls). When present, ENDO-I mRNA and protein were observed in the functionalis zone of endometrial stroma and the stroma of endometriotic lesions. Expression and localization differences were scored and statistically analysed. During the secretory stage, ENDO-I mRNA expression by endometriotic lesions and eutopic endometrium from women with disease was significantly greater than ENDO-I mRNA expression by proliferative stage eutopic endometrium from women with disease or eutopic endometrium from controls, regardless of cycle stage (P < 0.001). More ENDO-I protein was localized in endometriotic lesions and eutopic endometrium from women with disease than in eutopic endometrium from controls, regardless of cycle stage (P < 0.001). Differential expression and localization of ENDO-I may help develop minimally invasive diagnostic strategies for endometriosis. Further, as ENDO-I shares nucleotide sequence and amino acid sequence with hepatic haptoglobin-which in certain disease states is immunosuppressive and angiogenic-differences in ENDO-I expression and localization in the peritoneal cavity may contribute to the pathogenesis of endometriosis and/or facilitate development of unprecedented diagnostic or therapeutic approaches for management of this enigmatic disease.     

Immunohistochemical localization of androgen receptor in the human endometrium, decidua, placenta and pathological conditions of the endometrium.

The immunohistochemical localization of the androgen receptor in the human endometrium at various stages of the menstrual cycle and post-menopausal period, in decidua and placenta of early pregnancy, and in several pathological conditions of the endometrium has been investigated. At any phase of the menstrual cycle, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity. There was little staining in myometrium. Even after menopause, endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak. In decidua and placenta of early pregnancy, decidual and trophoblastic cells showed positive staining and there was no staining in the stromal cells of placenta. The expression of the androgen receptor was also detected in adenomyosis, endometriosis and endometrial carcinoma. Although the proliferation and differentiation of endometrium are mediated mainly by oestrogen and progesterone receptors, the androgen receptor may play some role in modulating these changes. These results suggest that it may be involved in both physiological and pathological changes of the endometrium.     

Oestrogen and progesterone receptor immunocytochemistry in human hyperplastic and neoplastic endometrium.

Proliferative disorders of the endometrium may be associated with autocrine and paracrine actions between stromal and epithelial cells. To determine whether the stromal-epithelial relation with respect to oestrogen receptor (OR) and progesterone receptor (PR) is disturbed in (pre)malignant endometrium immunocytochemical OR and PR expression was quantitated by computerized image analysis. This was studied in the stromal and epithelial cells of endometrial specimens diagnosed as hyperplasia (n = 14), atypical hyperplasia (n = 16), and adenocarcinoma (n = 33). Paraffin sections were used for optimal preservation of histomorphology. A progressive loss of OR and PR content occurred with increasing malignant transformation. Stromal cells in atypical hyperplasia (P = 0.0007) and well-differentiated adenocarcinoma (P = 0.0008) exhibited a relative loss of PR content as compared with epithelial cells (P = 0.036 and P = 0.17, respectively). In atypical hyperplasia, the decrease in stromal PR content was not in parallel with persistent stromal OR immunostaining. Furthermore, stromal PR expression in atypical hyperplasia was significantly (P = 0.004) lower than in the surrounding hyperplasia, whereas the stromal OR staining as well as the epithelial OR and PR staining did not differ significantly. These observations may reflect a disturbance in hormonal interrelationships between endometrial cells in the development of endometrial neoplasia, indicating that stroma may modulate epithelial growth by paracrine mechanisms.     

Aberrant expression of beta-catenin and mutation of exon 3 of the beta-catenin gene in renal and urothelial carcinomas

The present study attempted to clarify the significance of aberrant expression of beta-catenin protein and mutation of exon 3 of the beta-catenin gene in renal and urothelial carcinogenesis. beta-Catenin expression was examined immunohistochemically and mutation of the beta-catenin gene was analyzed by polymerase chain reaction-single strand conformation polymorphism (SSCP) and direct sequencing. beta-Catenin immunoreactivity was observed at the cell membrane in all 30 renal cell carcinomas (RCC) examined, and no RCC showed a mobility-shifted SSCP band. Of 46 transitional cell carcinomas (TCC) examined, there was reduced expression of beta-catenin, as compared with its expression in non-cancerous transitional epithelium, in 22 cases (48%) and beta-catenin accumulation in the nucleus in five cases (11%). Of four renal pelvis TCC examined, point mutation of exon 3 of the beta-catenin gene at codon 45 resulting in amino acid substitution (Ser to Phe) was detected in one (25%). The incidence of reduced expression of beta-catenin correlated significantly with the growth pattern (superficial type vs invasive type) of TCC (P < 0.05). These data indicate that: (1) aberrant beta-catenin expression may be at least partly involved in urothelial carcinogenesis, but less significantly so in renal carcinogenesis, and (2) it may be associated with the progression of TCC showing invasive growth.     

Studies on the intranuclear localization of influenza virus-specific proteins.

The major influenza virus-specific proteins appearing in the nucleus of infected cells are the virion nucleocapsid protein (NP) and a putative nonstructural protein (NS₁) of approximately 25,000 daltons. In the present study, we have determined the intranuclear localization of NP and NS₁ in two cell lines, canine kidney cells (MDCK cells) and HeLa cells. Nuclear fractionation was monitored by using ribosomal precursor RNA and heterogeneous nuclear RNA as markers for nucleolar and nucleoplasmic components, respectively; and nucleoli were purified by equilibrium centrifugation in Renografin gradients.The purified nucleoli contained essentially no NP protein, indicating that this protein is mostly, if not totally, nucleoplasmic in location. Some of the nuclear NS₁ remained associated with the purified nucleoli. Of the NS₁ in the nucleus of infected HeLa cells, 35% remained with the nucleoli, whereas in MDCK cells only 6% remained. Reconstruction experiments further suggest that the NS₁ found tightly associated with nucleoli did not arise as an artifact of nuclear fractionation.     

The proapoptotic factor HLDF in the normal, hyperplastic and neoplastic endometrium

Antibodies to the factor HLDF are shown to be specific markers of apoptosis and permit the estimation of the rate of programmed cell death in the course of a normal menstrual cycle and in pathologic endometrial processes. HLDF expression in the epitheliocytic cytoplasm makes it possible to evaluate apoptosis at early stages, before the emergence of the first morphological signs and after apoptotic body formation. The study shows increased apoptotic processes at the end of a normal menstrual cycle and during neoplastic cell transformation. Antibodies to the HLDF factor may be used as a new immunohistochemical marker for the differential diagnosis of benign and malignant endometrial processes.     

Silver-stained nucleolar organizer regions in the normal,hyperplastic and neoplastic endometrium

Summary A morphometric evaluation of number and grouping of megakaryocytes (MK) in five different groups of chronic myeloproliferative disorders (CMPD) was performed by counting 60 high power fields equaling approximately 14.28 mm² of haematopoiesis in each case. Twenty-one up to 29 cases were evaluated for each of five categories of CMPD and one control group; a total of 132 cases of CMPD and 33 control cases were used. The mean number of MK per square millimetre was 15.54±1.53 in chronic myeloid leukaemia of common or granulocytic type (CML.CT), 69.91±5.85 in CML with megakaryocytic increase (CML.MI), 59.59 ±3.27 in polycythaemia vera (P. vera), 59.85±4.59 in primary thrombocythaemia (PTH), 67.58±4.11 in chronic megakaryocytic granulocytic myelosis (CMGM), and 19.7±3.07 in controls. The distinction between free or isolated MK, and between clustered or grouped MK corresponds to the total cell counts of MK in the various groups of CMPD. Clustering of MK was significantly higher in CMGM and PTH compared to other groups, but the difference between them was not statistically significant. Significant differences in the mean number of MK were obtained between controls and CML.CT on the one hand and all other groups of CMPD on the other. The results further support the histological sub-classification of CMPD according to the primary disorders of the Hannover classification (not advanced by sclerosis, fibrosis or excess of blasts, respectively).     

Adrenomedullin, Bcl-2 and microvessel density in normal, hyperplastic and neoplastic endometrium

Adrenomedullin (ADM) is a multifunctional 52-amino acid peptide involved in numerous physiological and pathological processes, including angiogenesis, growth regulation, differentiation, and vasodilation. ADM is thought to act through the G protein-coupled receptor calcitonin receptor-like receptor, with specificity being conferred by receptor-associated modifying protein 2. The aim of the present study was to clarify the roles of ADM status, and tumor vessels in endometrium. Specimens were examined for ADM, microvessel density (MVD), area of venules (AV) and Bcl-2 oncoprotein using an immunoperoxidase method. The difference of ADM between normal proliferative phase and hyperplasia without atypia was significant ( P < 0.05). The level of Bcl-2 was significantly different between hyperplasia without atypia and hyperplasia with atypia ( P < 0.05). ADM, MVD and AV in the endometrium increased in a stepwise manner from normal, simple or complex hyperplasia with or without atypia to grade 1 adenocarcinoma. In contrast, expression of Bcl-2 oncoprotein was decreased. These parameters identify the role of ADM expression and Bcl-2 protein in relation to cell growth and vasodilating in the neoplastic changes.     

Spontaneous neoplastic lesions in Harlan Sprague-Dawley rats.

In a 24-months study, the spontaneous tumour spectrum of the Hsd:Sprague-Dawley stock was examined. Pituitary gland tumours were found in 20% of the males and 39% of the females. This relatively low incidence, compared to other SPRD stocks, had little effect on the survival of females (50%), due to the high incidence (76%) of mammary gland tumours (predominantly fibroadenomas) that resulted in unscheduled sacrifices of many females. Other common neoplasms in Hsd:Sprague-Dawley rats were benign medullary tumours (27% in males, 11% in females), C-cell adenomas (23% in males, 28% in females), and endometrial stromal polyps (22% in females).     

Estrogen metabolizing enzymes in endometrium and endometriosis

BACKGROUND: Estradiol (E(2)) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS: In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17beta-hydroxysteroid dehydrogenases (17beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS: Aromatase and 17beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17beta-HSD, EST and STS were readily detectable. Only 17beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS: In endometriosis lesions, the balance is tilted in favor of enzymes producing E(2). This is due to a suppression of types 2 and 4 17beta-HSD, and an increased expression of aromatase and type 1 17beta-HSD in ectopic endometrium.     

DNA-flow-cytometric measurements on the normal,atrophic, hyperplastic and neoplastic human endometrium

Summary DNA distribution patterns and the fractions of the cell cycle phases were determined by means of flow-through cytometry in 87 samples of normal, atrophic, hyperplastic and carcinomatous human endometrium. The S-phase fractions vary during the normal menstrual cycle between 1 and 3% and reach a periovulatory maximum between 4.4 and 4.7%. Atrophic endometrium and regressive-glandular cystic hyperplasia have little DNA synthesis (1.01% and 1.68% S-phase fractions respectively). Proliferating glandular cystic hyperplasia reveals 3.38% S-phase fraction, whereas adenomatous hyperplasia has an increased number of DNA-synthesizing cells (4.81%). The well-differentiated endometrial carcinoma shows no cytophotometrically detectable differences in comparison to adenomatous hyperplasia. All endometrial samples except for poorly differentiated endometrial carcinoma showed a diploid to tetraploid DNA distribution pattern. The poorly differentiated endometrial carcinoma displays two different types: one rapidly growing diploid-tetraploid tumor with 8.0 to 9.6% S-phase fractions, and another type with stemline deviations, polyploid nuclei and less pronounced synthetic activity.Dedicated to Professor Volker Becker at the occasion of his 60th anniversary     

E-cadherin promoter hypermethylation in preneoplastic and neoplastic skin lesions.

E-cadherin is a calcium-dependent, intercellular adhesion molecule that is specifically expressed in epithelial tissues and plays an important role in maintaining epithelial stability. E-cadherin is widely regarded as a prognostic marker in many types of human cancers. The inactivation of the E-cadherin gene is linked to increased potential for tumor invasiveness and distant metastasis. We previously demonstrated reduced expression of E-cadherin protein immunohistochemically in invasive squamous cell carcinomas of the skin as compared with adjacent normal skin. An epigenetic alteration in association with promoter hypermethylation is one important mechanism of gene silencing. In the present study, we analyze the E-cadherin gene promoter hypermethylation in preneoplastic and neoplastic skin lesions to determine whether epigenetic alteration of the E-cadherin gene also plays an important role in cutaneous squamous carcinogenesis. A total of 33 cases was examined for evidence of E-cadherin promoter hypermethylation, and these consist of nine cases of spongiotic dermatitis as nonneoplastic skin control, nine cases of actinic keratosis, eight cases of squamous cell carcinoma in situ, and seven cases of invasive squamous cell carcinoma. Promoter hypermethylation of the E-cadherin gene was detected in 6 of 7 cases (85%) of invasive squamous cell carcinoma, 4 of 8 cases (50%) of squamous cell carcinoma in situ, 4 of 9 cases (44%) of actinic keratosis, and 2 of 9 cases (22%) of nonneoplastic skin. We conclude that E-cadherin promoter hypermethylation occurs frequently and may represent an important mechanism of E-cadherin inactivation in cutaneous preneoplastic and neoplastic lesions. The frequencies of E-cadherin promoter hypermethylation appear to be correlated with more advanced stage of squamous carcinogenesis in skin.     

Cellular localization and signaling activity of beta-catenin in migrating neural crest cells.

In the vertebrate embryo, development of the neural crest is accompanied by sequential changes in cellular adhesiveness, allowing cells to delaminate from the neural epithelium, to undergo migration through extracellular matrix material, and to coalesce into ganglia of the peripheral nervous system. Because of its dual role in cell adhesion, as a link between cadherins and the actin cytoskeleton, and in cell signaling, as a key mediator of the Wnt-signaling pathway, beta-catenin is a good candidate to play a central role in the control of neural crest cell development. In the present study, we analyzed, by using an in vitro culture system, whether the cellular localization and the signaling activity of beta-catenin are regulated in conjunction with cell migration during ontogeny of trunk neural crest cells in the avian embryo. beta-Catenin molecules were found primarily in association with N-cadherin in the regions of intercellular contacts in most migrating neural crest cells, and only early-migrating cells situated in proximity with the dorsal side of the neural tube showed detectable beta-catenin in their nuclei. This finding indicates that beta-catenin may be recruited for signaling in neural crest cells only transiently at the onset of migration and that sustained beta-catenin signals are not necessary for the progression of migration. The nuclear distribution of beta-catenin within crest cells was not affected upon modification of the N-cadherin-mediated cell-cell contacts, revealing that recruitment of beta-catenin for signaling is not driven by changes in intercellular cohesion during migration. Overstimulation of beta-catenin signals in neural crest cells at the time of their migration, using LiCl treatment or coculture with Wnt-1-producing cells, induced nuclear translocation of beta-catenin and Lef-1 up-regulation in neural crest cells and provoked a marked inhibition of cell delamination and migration. The effect of LiCl and exogenous Wnt-1 on neural crest cells could be essentially attributed to a dramatic decrease in integrin-mediated cell-matrix adhesion as well as a massive reduction of cell proliferation. In addition, although it apparently did not affect expression of neural crest markers, Wnt-1 exposure dramatically affected signaling events involving Notch-Delta, presumably also accounting for the strong reduction in cell delamination. In conclusion, our data indicate that beta-catenin functions primarily in cell adhesion events during migration and may be recruited transiently for signaling during delamination possibly to regulate the balance between cell proliferation and cell differentiation.     

Significance of hormone receptor status and tumor vessels in normal, hyperplastic and neoplastic endometrium

The aims of this study were to identity the roles of tumor vessels and hormone receptor status in normal, hyperplastic, and neoplastic endometrium, and to explore their relationships with other prognostic factors of endometrial adenocarcinoma. Endometrial curettage specimens of proliferative phase and secretory phase endometrium, simple hyperplasia with or without atypia, complex hyperplasia with or without atypia, and grade 1 adenocarcinoma were examined for estrogen receptor alpha (ER alpha), progesterone receptor (PgR), Ki-67 labeling index (LI), cyclin D1, microvessel density (MVD), and area of venules (AV) using an immunoperoxidase method. The results showed high levels of ER alpha in complex hyperplasia, and high levels of PgR in simple hyperplasia without atypia. Expression of ER alpha in the endometrium decreased in a stepwise manner from complex hyperplasia without atypia to grade 1 adenocarcinoma. Expression of PgR in the endometrium decreased in a stepwise manner from simple hyperplasia without atypia to grade 1 adenocarcinoma. In contrast, the expressions of Ki-67 LI, cyclin D1, MVD and AV in the endometrium increased in a stepwise manner from normal, simple or complex hyperplasia with or without atypia to grade 1 adenocarcinoma. These changes may become irreversible on progression from simple or complex hyperplasia to neoplasia.     

Analysis of beta-catenin mutations and alpha-, beta-, and gamma-catenin expression in normal and neoplastic human pituitary tissues

The cadherin-catenin system mediates Ca²⁺-dependent cell-cell adhesion, and genetic alterations in these molecules play a significant role in multistage carcinogenesis. Mutations in the β-catenin gene, mostly affecting exon 3, have been detected in malignant cell lines and in primary tumors. Immunohistochemical abnormalities in α-, β-, and γ-catenin have been reported in malignant and benign tumors, and nuclear localization of β-catenin has been associated with mutations in exon 3 of this gene. Mutational analysis of exon 3 of the β-catenin gene was undertaken by polymerase chain reaction (PCR) and sequencing using genomic DNA extracted from frozen tissues, including 4 normal pituitaries, 22 pituitary adenomas, and one pituitary carcinoma. Frozen sections from these cases were used for immunohistochemical detection of β-catenin. We also analyzed immunohistochemical expression of α-, β-, and γ-catenin by paraffin sections from 154 pituitary tumors, including 148 adenomas and 6 carcinomas. Genomic DNA was extracted from paraffin sections of 2 gonadotroph tumors showing nuclear staining for β-catenin and was used for PCR and sequencing of exon 3 of the β-catenin gene. No mutations in exon 3 of the β-catenin gene were found in any of the 23 cases analyzed by PCR and sequencing. In addition, the 2 cases studied by paraffin section immunohistochemistry, with nuclear staining for β-catenin, were negative for mutations in this exon. Normal pituitary expressed all three catenin proteins. Immunostaining usually showed a membranous pattern of reactivity and was generally stronger in normal pituitary than in the adjacent adenomas. Stains for α-catenin were positive in fewer tumors than for β-catenin. The lowest frequency immunopositive tumors and the weakest immunostaining was for γ-catenin. All medically treated prolactinomas were negative for γ-catenin, whereas treated growth hormone adenomas were less often positive for both α- and γ-catenin than for untreated tumors. The percentage of positive cases for β-catenin was the same in these two groups. Most pituitary carcinomas were negative for both α- and γ-catenin but were β-catenin positive. These results indicate that (i) mutations in exon 3 of the β-catenin gene are uncommon in pituitary tumors, and (ii) expression of α-, β-, and γ-catenin is decreased in pituitary adenomas compared to normal pituitary tissues.     

Immunolocalization of cystatin A in neoplastic, virus and inflammatory lesions of the uterine cervix.

Cystatin A was immunohistochemically demonstrated in the normal squamous epithelium of the uterine cervix, particularly in the parabasal and superficial cell layers whereas it was absent or scanty in the basal cells and in areas with parakeratosis. Cystatin A was also found in neoplastic lesions (dysplasia, carcinoma in situ and squamous cell carcinoma), but less abundant than in normal squamous epithelium. The immunoreaction in intraepithelial neoplasia was closely related to the degree of morphological maturation of the squamous cells with more abundant cystatin A in low grade dysplasia and less in high grade dysplasia and carcinoma in situ. In squamous cell carcinoma, cystatin A was often abundant in highly differentiated areas and almost absent in poorly differentiated ones. Cystatin A was found in the squamous epithelium in herpes and in condylomatous lesions. It was also found in the cytoplasm of neutrophils, but not in lymphocytes and plasma cells. In unspecific cervicitis, cystatin A was found extracytoplasmatically as small vesicles in the epithelial-stromal junction. The implications of cystatin A in neoplastic, virus, and inflammatory processes are discussed.     

Protoplast formation and localization of enzymes in Streptococcus mitis.

Cells of Streptococcus mitis ATCC 903 were converted to stable protoplasts by the cell wall-degrading M-1 enzyme of the mutanolysin complex isolated from Streptomyces globisporus. Over 90% of total glucokinase (EC 2.7.1.2), aminopeptidase (EC 3.4.11.1), and dextranglucosidase (EC 3.2.1.70) was recovered in the cytoplasmic fraction, whereas over 20% of total invertase (beta-fructofuranosidase: EC 3.2.1.26) was released during protoplast formation. ATPase (EC 3.6.1.3). chymotrypsin-like protease (EC 3.4.21.1), arginine aminopeptidase (EC 3.4.11.6), and lactate dehydrogenase (EC 1.1.1.27) were detected in Triton X-100 extracts of the cytoplasmic membrane fraction by crossed immunoelectrophoresis in combination with enzyme-staining procedures. By these methods, NADH dehydrogenase (EC 1.6.99.3), aminopeptidase, and lactate dehydrogenase were detected in the cytoplasmic fraction. Aminopeptidases in the cytoplasmic fraction differed from this activity in the membrane fractions in electrophoretic mobility and substrate specificity.