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PubMed1990

The collagen fibril--a model system for studying the staining and fixation of a protein.

A collagen fibril is made up of long rod-like molecules regularly D-staggered with respect to one another. This means that (i) its axially projected fine structure, resolvable to approximately 2 nm in electron micrographs, repeats D-periodically (D = 67 nm), and (ii) the amino acid residues contributing to each element of the fine structure can be inferred from sequence data. Electron-optical data from a fibril D-period can can therefore be correlated directly with chemical data. Such correlations confirm the electrostatic nature of the staining reaction when a fibril is positively stained. After negative staining, the principal factor determining the small-scale distribution of stain is local exclusion by 'bulky' amino acid side-chains. ('Bulkiness' is the average cross-sectional area, or 'plumpness', of a side-chain.) A small superimposed positive staining contribution can also be detected. Fixation of collagen by aldehydes and diimidoesters occurs via an initial reaction with lysyl (and hydroxylsyl) side-chains and alpha-amino groups, followed by secondary cross-linking reactions that differ from fixative to fixative. These secondary reactions determine the nature and abundance of the cross-links and the extent to which they influence subsequent staining behaviour.

Amino Acid Sequence↗
PubMed2000

Basal cell adenocarcinoma of the lacrimal gland.

PURPOSE: Basal cell adenocarcinoma is a recently recognized entity among malignant tumors of the salivary glands. This entity has not received enough attention among ophthalmologists and ophthalmic pathologists. We recommend that basal cell adenocarcinoma be included in the differential diagnosis of lacrimal gland tumors. DESIGN: Case report. METHODS: In 1988 a lacrimal gland tumor was incompletely removed from the left orbit of a 36-year-old woman and diagnosed as "solid basaloid adenoid cystic carcinoma". Soon after, an exenteration of the orbit was performed. In 1998, upon review of the initial histopathology, the diagnosis of solid adenoid cystic carcinoma was changed to basal cell adenocarcinoma. RESULTS AND MAIN OUTCOME MEASURE: The pathologic findings included nests of basaloid cells with minimal atypia and incomplete palisading around the periphery. The cells were of two types. The first type were large, pale cells with round or oval nuclei, scanty cytoplasm, and ill-defined borders. The other type were smaller cells with hyperchromatic nuclei situated mainly near the periphery of the nests. There was no necrosis or perineural invasion. Mitotic figures were present. Cysts within the nests showed Alcian blue negative contents. Immunohistochemistry showed a positive reaction to cytokeratin and a negative reaction with smooth muscle actin (SMA). CONCLUSIONS: The Alcian blue negative stain, the negative reaction to SMA, and the fact that the patient is still alive 10 years after exenteration favored the diagnosis of basal cell adenocarcinoma. It is essential to differentiate a basal cell adenocarcinoma of the lacrimal gland from the solid basaloid type of adenoid cystic carcinoma, because the former has a better prognosis. Patients with basal cell adenocarcinomas of the lacrimal gland should be closely monitored for local recurrences because this tumor has the tendency to show perineural invasion.

Adenocarcinoma↗
PubMed2004

Diagnosis and reporting of limited adenocarcinoma of the prostate on needle biopsy.

The diagnosis of limited adenocarcinoma of the prostate is one of the more difficult challenges in surgical pathology. This paper highlights the methodological approach to diagnosing limited cancer, based on a constellation of features more commonly present in adenocarcinoma than benign glands. In assessing small foci of atypical glands on needle biopsy, one looks for differences between the benign glands and the atypical glands in terms of nuclear features, cytoplasmic features, and intraluminal contents. Only a few features, such as glomerulations, mucinous fibroplasia (collagenous micronodules), and perineural invasion are diagnostic in and of themselves for prostate cancer. Immunohistochemistry may be a useful adjunct in the diagnosis of limited adenocarcinoma of the prostate, although as with any immunohistochemical studies, there are problems with both sensitivity and specificity. Basal cell markers, such as high molecular weight cytokeratin and more recently, p63, highlight basal cells found in benign glands, yet are absent in adenocarcinoma of the prostate. However, not all benign glands label uniformly with basal cell markers. Certain mimickers of adenocarcinoma of the prostate are even less frequently labeled uniformly with these stains. Consequently, negative staining in a small focus of atypical glands for basal cell markers is not diagnostic of adenocarcinoma of the prostate. More recently, a marker has been identified that relatively selectively labels adenocarcinoma of the prostate. AMACR will label the cytoplasm of approximately 80% of limited adenocarcinoma of the prostate cases on needle biopsy. In positive cases, not all of the glands will be positive and those that are positive are often not intensely positive. Certain variants of adenocarcinoma of the prostate that are a little more difficult to recognize, such as foamy glands adenocarcinoma, pseudohyperplastic adenocarcinoma, and atrophic adenocarcinoma, are labeled with AMACR in only approximately 60-70% of cases. In addition to problems with sensitivity, AMACR is not entirely specific for adenocarcinoma, and will label almost all cases of high-grade prostatic intraepithelial neoplasia, some foci of adenosis, and even some entirely benign glands. Finally, this paper will briefly cover the significance of atypical or suspicious prostate needle biopsies, and how to report the key diagnostic and prognostic information on needle biopsy.

Adenocarcinoma↗
PubMed1998

Incidence of apoptosis and metallothionein expression in renal cell carcinoma.

OBJECTIVE: To investigate the frequency of apoptosis and expression of metallothionein (MT) protein and to clarify their roles in renal cell carcinoma (RCC). MATERIALS AND METHODS: Apoptosis was detected by using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labelling (TUNEL) technique in 70 formalin-fixed and paraffin-embedded RCC specimens. The expression of MT was determined immunohistochemically. RESULTS: The incidence of apoptosis was significantly higher in grade 2 and 3 RCC than in grade 1. There were no significant differences amongst the other categories of RCC when grouped by tumour stage, cell type and growth pattern. The expression of MT was detected in 23 of 70 (33%) RCCs. Subcellularly. MT was localized in the cytoplasm, nucleus and cell membrane of RCC. Statistical analysis showed a close association of MT expression with the higher grades and invasive growth pattern of RCC. In addition, the incidence of apoptosis increased with increasing immunoreactivity of MT staining. There was a significant difference in the incidence of apoptosis between the diffuse staining and negative staining of MT. CONCLUSIONS: There were close associations of higher grade RCC with both the incidence of apoptosis and the expression of MT, as well as a close relationship between MT expression and the invasive growth pattern of RCC. This suggests that the involvement of apoptotic cell death and MT expression should be considered an important factor in the process of the renal carcinogenesis and tumour progression.

Adult↗
PubMed2002

Cutaneous leishmaniasis: Texas case diagnosed by electron microscopy.

A 78-year-old woman from south-central Texas without a history of travel outside the state presented with an erythematous papule on her right forearm. A biopsy specimen showed non-necrotizing granulomatous inflammation with negative stains for organisms. The lesion was recalcitrant to treatment with topical steroids, and a second biopsy specimen, obtained 3 months later, showed a histiocytic infiltrate in the upper dermis. Numerous, round, 1- to 30-microm bodies were seen within vacuoles in the histiocyte's cytoplasm. Giemsa, Grocott-Gomori methenamine-silver nitrate, and periodic acid-Schiff stains were negative. Electron microscopic examination of the paraffin-embedded tissue showed parasites diagnostic of leishmaniasis. On review of the literature, we found that 29 other cases of autochthonous cutaneous leishmaniasis have been reported in Texas. We emphasize the endemic nature of this condition in Texas.

Aged↗
PubMed2005

INI1 protein expression distinguishes atypical teratoid/rhabdoid tumor from choroid plexus carcinoma.

Central nervous system atypical teratoid/rhabdoid tumor (AT/RT) and choroid plexus carcinoma (CPC) are rare, highly malignant tumors that predominantly arise in infants and young children. Overlapping clinical, histologic, ultrastructural, or immunophenotypic features may obscure the diagnosis in some cases. AT/RT is characterized by deletions and/or mutations of the INI1 tumor-suppressor gene on chromosome band 22q11.2. We have recently developed an INI1 immunohistochemical staining assay. Negative staining of tumor cells resulting from inactivation of the INI1 gene is a consistent feature of AT/RT. Mutations of INI1 in some CPCs have been reported. The purpose of the present study was to determine if immunohistochemical staining with an INI1 antibody would provide a sensitive means of distinguishing between CPC and AT/RT. We examined 28 tumors with a submitted diagnosis of CPC. Twenty-one CPCs showed retained expression of INI1 and seven tumors showed loss of INI1 expression. Cytogenetic, FISH, and/or INI1 mutation results were also available for 13 tumors. In three of the seven cases, monosomy 22 was the only cytogenetic abnormality, suggestive of AT/RT. However, monosomy 22 was also identified in 3 tumors with complex karyotypes that retained INI1 expression. The 7 tumors that were immunonegative for INI1 had features that were consistent with AT/RT. Immunostaining for INI1 protein is retained in the majority of CPC and is lost in AT/RT. This expression pattern seems to better define the 2 groups of tumors than does light or electron microscopy, routine immunohistochemistry, or cytogenetic analysis alone.

Adolescent↗
PubMed2000

High-resolution ultrastructure of amyloid fibrils in familial amyloid polyneuropathy.

The ultrastructure of amyloid fibrils in familial amyloid polyneuropathy (FAP) was clearly demonstrated. Amyloid of three patients with FAP caused by the point mutation of the 30th amino acid of transthyretin (ATTR Val30Met) and one patient with FAP caused by two point mutations of the 30th and 104th amino acid of transthyretin (ATTR Val30Met/Arg104Cys) were partially isolated, stained negatively and examined with an electron microscope. Amyloid fibrils of both types were composed of two protofilaments and twisted at 180 degrees to the right and left alternately with a periodicity of 125-135 nm. This is the first report demonstrating such unique alternating twist structure of amyloid fibrils. There were no ultrastructural differences between the fibrils caused by the ATTR Val30Met and ATTR Val30Met/ Arg104His; therefore, it is suggested that the point mutation of the 30th amino acid of transthyretin might play an important role in the formation of amyloid fibrils. Further biochemical study on the mechanism of this alternating twist formation should be undertaken.

Amino Acid Substitution↗
PubMed2000

Staining of melanocytic neoplasms by melanoma antigen recognized by T cells.

We stained benign melanocytic nevi and malignant melanoma with antibodies to melanoma antigen recognized by T cells (Mart-1) to determine if this was useful in differentiating benign from malignant melanocytic neoplasms. Forty-five primary malignant melanomas and 71 benign melanocytic nevi were stained with antibodies to Mart-1. Two cases of malignant melanoma metastatic to lymph node and three cutaneous metastases of malignant melanoma were also stained. The degree of staining was graded into diffuse positive staining, focal positive staining, and negative staining. Thirty-six of 45 primary malignant melanomas stained diffusely positive with antibodies to Mart-1. This included three of five desmoplastic malignant melanomas that showed positive staining. Four melanomas showed faint or focal positive staining. One of two metastases to lymph node showed strong positive staining and one showed no staining. All three cutaneous metastases showed diffuse positive staining. Sixty-one of 71 melanocytic nevi showed no staining or faint staining with antibodies to Mart-1. Ten of 71 melanocytic nevi showed strong positive staining. The majority of these were congenital nevi. Staining with antibodies to Mart-1 antigen was a useful marker of malignant melanoma. However, staining may also be seen in benign melanocytic neoplasms. The presence or absence of staining for Mart-1 antigen cannot be used to differentiate benign melanocytic nevi from malignant melanocytic tumors.

Antigens, Neoplasm↗
PubMed2001

A prospective study of p53 expression and its correlation with clinical response of radiotherapy in nasopharyngeal carcinoma.

OBJECTIVES/HYPOTHESIS: Nasopharyngeal carcinoma (NPC) is a common malignant neoplasm of the head and neck that occurs in people in the southeastern Asian area, including Taiwan. The significant association of p53 expression in NPC suggested that p53 overexpression seemed to occur at an early stage in the development of NPC. Alterations of p53 status were probably the most commonly encountered in head and neck carcinomas, and there was extensive evidence that p53 status might determine tumor response to therapy. Ionizing radiation was studied extensively for the relationship between its damaging effect and p53 status in human cancer cells. STUDY DESIGN: This study was carried out to investigate whether there was any correlation between overexpression of p53 protein and locoregional tumor response in patients with NPC treated with 7000 cGy of radiotherapy. METHODS: Sixty-eight patients (50 males, 18 females) with NPC who were diagnosed and treated with radiotherapy were studied prospectively. Before they had received a radiation dose of 7000 cGy in 35 fractions, five fractions a week, p53 status from a nasopharyngeal biopsy was studied using immunohistochemical staining (IHC). RESULTS: The locoregional response rate of primary tumor was analyzed statistically. Forty-seven patients (69.1%) showed positive p53 staining in their tumors. There were 5 positive stains in 6 squamous cell carcinomas (SCC; 83.3%), 34 positive in 53 non-keratinizing carcinomas (NKC; 64.2%), and 8 positive in 9 undifferentiated carcinomas (UC; 88.9%). The mean ages for patients with three different histopathologies were 48.5, 46.1, and 61.1 years. There were 8 patients (7 positive stains, 1 negative stain) with residual tumor after radiotherapy and all were NKC (6 males, 2 females). Therefore, the clinical response rate of primary tumor was 85.1% in positive p53 immunostaining (40 of 47 cases), 95.2% in those with no immunostaining (20 of 21 cases); the former was poorer in locoregional tumor response than the latter, but there was no significant difference (P > .05, chi2 test). CONCLUSIONS: We conclude that there is no statistically significant correlation in locoregional response of primary tumor between p53 overexpression and radiotherapy in patients with NPC (P > .05, Fisher exact test).

Adolescent↗
PubMed2001

Anaerobranca gottschalkii sp. nov., a novel thermoalkaliphilic bacterium that grows anaerobically at high pH and temperature.

A novel thermoalkaliphilic, obligately anaerobic bacterium was isolated from a humid soil sample of a hot inlet of Lake Bogoria, Kenya. The newly isolated strain grows optimally at pH 9.5 and 50-55 degrees C and its growth range is pH 6.0-10.5 and 30-65 degrees C. Unlike the already known thermoalkaliphiles, the strain grows heterotrophically on a variety of mono- and polysaccharides (glucose, ribose, mannose, fructose, sucrose, maltose, starch, pullulan, xylan and cellulose) and on proteinaceous substrates such as yeast extract, peptone and tryptone. No dissimilatory sulfate reduction was observed, whereas thiosulfate was found to enhance growth when glucose or starch were used as substrates. Under optimal conditions, the doubling time is 48 min. Sodium ions are necessary for growth, with an optimal concentration of 230 mM (1% NaCl, w/v) at pH 9.5. The rod-shaped cells are motile in the exponential growth phase under optimal growth conditions. Despite the Gram-negative staining and negative KOH assay, the strain is a Gram-positive organism, having an atypically thin cell wall. A sheath-like structure occurs at the cell separation area and parts of a surface layer-like structure were also observed. Based on physiological properties and molecular biological analysis, the strain falls within the radiation of the clostridia and represents a new species of Anaerobranca within the Clostridium/Bacillus subphylum of the Gram-positive bacteria. Strain LBS3T (= DSM 13577T) is named Anaerobranca gottschalkii sp. nov. and is designated as the type strain.

Alkalies↗
PubMed1999

High-resolution ultrastructural comparison of renal glomerular and tubular basement membranes.

BACKGROUND/AIMS: Glomerular basement membranes (GBM) and tubular basement membranes (TBM) consist of a fine meshwork composed mainly of type IV collagen. Each segment of tubules has specialized physiologic functions, and thus we investigated the ultrastructure of various basement membranes in rat kidneys. METHODS: Since purifying basement membranes from different tubule segments is technically challenging, we employed tissue negative staining rather than conventional negative staining to compare the ultrastructures of proximal and distal TBM and GBM in normal rats. We also assessed the distribution of extracellular matrix components including type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes by immunohistochemistry. RESULTS: TBM and GBM of normal rats showed a fine meshwork structure consisting of fibrils forming small round to oval pores. Short- and long-pore diameters in proximal tubules were 3.3 +/- 0.5 and 3.9 +/- 0.6 nm, respectively, and in distal tubules 3.5 +/- 0.7 and 4.3 +/- 0.8 nm, respectively. For GBM the respective diameters were 2.5 +/- 0.5 and 3.0 +/- 0.5 nm. Immunohistochemical analysis showed no significant difference in distribution of extracellular matrix components between proximal and distal TBM. However, immunofluorescence scores of alpha1 chain of type IV collagen, fibronectin, and laminin were higher in the TBM than in the GBM. On the other hand, heparan sulfate proteoglycan was higher in the GBM. CONCLUSION: Ultrastructural differences in renal basement membranes may be related to differences in physiologic function in each segment.

Animals↗
PubMed1990

Non-Hodgkin's lymphoma: a histopathologic and immunohistochemical study of 79 cases.

Recently immunophenotyping has become a valuable tool in the diagnostic workup of malignant lymphoma. We classified 79 consecutive cases of non-Hodgkin's lymphoma experienced at our hospital during the last two years according to the Working Formulation and immunologically using MT1, UCHL1 and MB2 monoclonal antibodies. The results of this study are as follows: 1) four cases (5.1%) were low grade, 54 cases (68.4%) were intermediate grade, and 21 cases (23.3%) were high grade. The most common subtype was 'diffuse, mixed' type, 2) fifty cases (63.3%) showed T-cell phenotype and 14 cases (17.7%) showed B-cell phenotype. Immunophenotyping was impossible in 15 cases due to either double staining or negative staining. 3) the incidence of extranodal presentation was high (65.8%) and the most common extranodal site was the upper aerodigestive tract (29.1%) followed by the gastrointestinal tract (16.4%), and 4) MT1, UCHL1 and MB2 monoclonal antibodies are valuable markers of T- and B-cells in paraffin embedded tissue, enabling retrospective study. However, because these antibodies are not lineage specific, the results of immunostaining should be interpreted with caution.

Adult↗
PubMedDate not supplied

p53 and MDM2 protein expression in squamous cell carcinoma and neighboring dysplasia from the upper aerodigestive tract.

BACKGROUND: The identification of genetic changes in preinvasive epithelial lesions may provide markers for a better assessment of the progression potential. MATERIALS AND METHODS: p53, MDM2 and PCNA immunophenotypes were examined in 57 cases of squamous cell carcinoma (SCC) of the upper aerodigestive tract and the adjacent normal and dysplastic epithelia. RESULTS: PCNA index increased with increasing grade of dysplasia. p53 protein was expressed in 35% and MDM2 protein in 33% of SCCs. The p53 protein was expressed in 89% of mild and moderate and in 93% of severe dysplasia and carcinoma in situ adjacent to p53-positive SCCs. The MDM2 protein was expressed in 30% of mild and moderate and in 54% of severe dysplasia and carcinoma in situ adjacent to MDM2-positive SCCs. Preinvasive lesions adjacent to negative SCCs stained negative. CONCLUSIONS: p53 protein was detected more frequently in preinvasive lesions than MDM2 protein and seems to be of greater value as a biomarker.

Adult↗
PubMed1992

Nonfunctioning pituitary adenoma.

A total of 14 patients (8 males and 6 females) with nonfunctional pituitary adenomas were diagnosed and underwent surgical intervention at the Kaohsiung Medical College Hospital between 1986 and 1991. Their ages ranged from 20 to 68 years with an average of 49.7 years. Eight adenomas were composed of small polygonal cells with chromophobic cytoplasm and other six were of large cells with pale eosinophilic granular cytoplasm. Immunohistochemical staining had negative staining of prolactin (PRL), growth hormone (GH), adrenal corticotropic hormone (ACTH) and thyroid stimulating hormone (TSH) in all cases, but scattered or wide spread cells containing alpha-subunit of glycoprotein hormones were found in 6 cases. Hormonal hypersecretion was absent in all of the clinical and biological pictures, as was reactivity to immunohistochemical examination. There were either very few signs of secretory activity, or none at all. CT and MRI scanning revealed 13 macroadenomas (92.9%) and one microadenoma. In seven cases, local invasion or suprasellar extension resulted in decreased visual acuity, but the most common clinical symptoms were progressive headache (78.6%), and visual disturbance (50.0%). After surgical intervention, symptoms disappeared in 74.2% of the patients.

Adenoma↗
PubMed1991

Clinical use of immunoperoxidase markers in excluding ectopic gestation.

Ectopic pregnancy is suspected when a pregnancy test is positive and an Arias-Stella phenomenon with decidua is seen on endometrial curettings in the absence of chorionic villi and trophoblast. We performed immunoperoxidase stains for beta-hCG, hPL, placental alkaline phosphatase, and keratin (AE1/AE3) on endometrial curettings of 72 patients with first-trimester bleeding in whom ectopic pregnancy was suspected based on routine hematoxylin and eosin (H&E) stain. Negative immunoperoxidase stains correlated with a retrospective patient chart review identified only ten patients with extrauterine pregnancy, a prevalence of 14%. The sensitivity of hCG, hPL, placental alkaline phosphatase, and keratin markers in detecting trophoblastic cells in the curettings was 42, 48, 24, and 69%, respectively. Each marker had a specificity of 100%. When both keratin and hPL were used, the sensitivity improved to 73%; however, there was no advantage in using three or more markers. We conclude that 73% of patients suspected to have ectopic pregnancy would be spared additional diagnostic techniques when immunoperoxidase markers for keratin and hPL are used on endometrial curettings without trophoblast on routine H&E stain. The procedure requires only 3 hours of laboratory time.

Decidua↗
PubMed2004

Negative 34betaE12 staining in a small focus of atypical glands on prostate needle biopsy: a follow-up study of 332 cases.

Atypical glands on prostate needle biopsy with a negative 34betaE12 (cytokeratin 903; CK903) immunostain, indicating a lack of a basal cell layer, are typically diagnostic of prostate cancer. However, in certain cases a negative 34betaE12 immunostain in a small focus of atypical glands is still not convincing enough to make the diagnosis of cancer. This study is the first report to evaluate the incidence of prostate cancer on follow-up biopsy in individuals with this diagnosis. A total of 543 men who had prostate core biopsy specimens diagnosed as a small focus of atypical-appearing glands with a negative 34betaE12 immunostain between January 1, 1997 and December 31, 2000 were selected for study. Some 61% of these 543 individuals (n = 332) had undergone at least one follow-up biopsy procedure. Of these, 43% of repeat biopsy cases (n = 142) were diagnostic of prostate cancer. A total of 46 individuals had at least 2 follow-up biopsy procedures, with 48% of these (n = 22) being diagnosed as cancer. The Gleason grades of the detected carcinomas were broken down as follows: Gleason grade 3 + 2 = 5, 6%; grade 3 + 3 = 6, 86%; grade 3 + 4 = 7, 1%; grade 4 + 3 = 7, 4%; and grade 4 + 4 = 8, 3%. The median amount of time to the first follow-up biopsy was 79 days, with 52% of follow-up biopsies performed within 90 days. A negative 34betaE12 immunohistochemical stain in a small focus of atypical glands is not associated with an increased prediction of prostate cancer on follow-up biopsy (43%), compared with previously published data for "small focus of atypical glands" alone (approximately 45%). Because 48% of men with an initial negative biopsy and multiple follow-up biopsy procedures were found to have cancer, more than one repeat biopsy session or more extensive sampling on the first repeat biopsy procedure may be necessary to maximize the identification of cancer. This finding is similar to that found in men with atypical diagnoses in general, without a negative 34betaE12 immunohistochemical stain. Only half of all individuals with a diagnosis of 34betaE12-negative focus of atypical glands underwent repeat biopsy within 3 months. Urologists need to be educated as to the significance of an atypical diagnosis and the need for repeat biopsy. In a small focus of atypical glands on prostate biopsy, negative staining for 34betaE12 should not necessarily lead to a definitive malignant diagnosis in all cases, because almost half of these biopsies on follow-up sampling are benign.

Adenocarcinoma↗
PubMed1979

Phase and amplitude contrast in electron microscopy of stained biological objects.

For biological objects negatively stained with heavy atom material, electron microscope images show best contrast for image detail on the scale of 10--20 A when a small objective aperture is used. In images taken under the optimum phase contrast imaging conditions of Scherzer, the required image detail is lost in unwanted noise. Both of these conditions may be described in terms of phase contrast imaging for a thin phase object. Calculations of image intensities and noise are reported for a model object consisting of heavy and light atoms randomly distributed to simulate a negatively stained protein molecule. The results are consistent with experimental observations.

Fourier Analysis↗
PubMed1984

Permeability changes in isolated liver mitochondria during different metabolic states.

The different staining patterns observed in isolated rat liver mitochondria suspended in a negative stain (2.11% ammonium molybdate) that were reported (U. Muscatello, V. Guarriera-Bobyleva, and P. Buffa, 1972, J. Ultrastruct. Res. 40, 235-260) were analyzed with the aim of determining the location of the stain within the mitochondria. The observations made by Muscatello and coworkers on whole, negatively stained mitochondria were confirmed. By embedding the negatively stained mitochondria according to one low denaturation embedding technique, the location of the stain could be determined. In rat liver mitochondria, freshly isolated, the stain was located within wide intracristal spaces. During state 4 respiration, stain was present in the cristae although the two membranes of each cristae were in most cases closely apposed. After 30 sec in state 3 respiration, the stain was present in the matrix, but absent from the cristae. During all three experimental conditions, stain was present in a narrow zone inside the membrane at the surface of the mitochondrion. The changes in permeability leading to an entrance of the stain into the matrix but not the cristae during state 3 respiration, and the reverse situation during state 4 shows that there are two pathways along which the stain can enter the matrix or the cristae. The permeability of these pathways is controlled independently. These observations cannot be explained on the basis of the generally accepted structure of mitochondria. The absence of any other structural modifications and a change in volume of the mitochondria in association with the transition from state 4 to state 3 respiration does not conform with the concept (C. R. Hackenbrock, 1966, J. Cell Biol. 30, 269-297) that the mitochondria undergo extensive "conformational" changes under these conditions.

Animals↗