Polyamine cytochemistry

Summary o-Phtalaldehyde (OPT) reacts with a number of biologically important molecules, including the polyamines, spermidine and spermine. By systematically varying reaction conditions with respect to temperature, pH, concentration and length of exposure to the reagent, using both model systems and tissues, we have succeeded in constructing a cytochemical OPT-method specific for spermidine and spermine. The method detects cell types known to contain these polyamines, including growing and neoplastic cells. The staining pattern obtained with the OPT method is identical to that obtained with the formaldehyde-fluorescamine (FF) technique recently shown to be specific for spermidine and spermine. In contrast to the FF technique, the OPT method can be used for staining suspensions of isolated cells and may hence be employed in studies using fluorescence-activated cell sorting (FACS). Preliminary such studies show a pronounced decrease in cellular OPT-induced fluorescence, paralleled by a decrease in content of polyamines, after treatment with the polyamine biosynthesis inhibitor -difluoromethylornithine (DFMO). In contrast, cells simultaneously treated with DFMO+spermidine show pronounced increases in their spermidine content and parallel increases in their OPT-induced fluorescence. Availability of methods selectively demonstrating polyamines at the cellular and subcellular level is expected to aid our understanding of polyamine functions in normal growth and cancer.     

Prospects for microscopical cytochemistry

Synopsis This review is devoted mainly to an evaluation of the status of microscopical cytochemistry seen as a discipline aiming at both the localization and the quantification of molecular processes in cells. Its relationships to ultramicrochemistry, as well as, in a broader sense, to biochemistry and cell biology, are discussed from both the historical and the methodological points of view. Recent developments in quantitative cytophysical techniques, such as automated cytophotometry using microscopes fitted with flying spot systems, TV cameras, or scanning stages, and the development of rapid flow cytometers are discussed. Analytical electron microscopy is touched upon too.The main part of the review is devoted to recent trends that strengthen the analytical basis of cytochemical staining methods. The special character of staining procedures as a kind of matrix chemistry is discussed and the potentialities of the use of matrixincorporated compounds for the fundamental study and calibration of microscopical staining procedures are elaborated. Parallel developments in the theory and practice of matrix chemistry in biochemistry are stressed. Growing interrelations between microscopical cytochemistry and related fields of investigation, such as the controlled fragmentation of cells, and methods like ultramicroanalysis of individual cells are indicated.     

Freeze-fracture cytochemistry: a new method combining immunocytochemistry and enzyme cytochemistry on replicas.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.     

Problems of cytochemistry of leukocytes]

In investigating the atypical granula in myeloic cells cytochemically the positivity of DMAB reaction identifies the specific eosinophilic nature of these granula. The slightly modified methods of representing histones allow those histones which are rich of lysine and arginine to be differentiated. With their help those differences can be grasped which exist between the condensed chromatin of lymphocytes and the mature neutrophils caused by the presence of histones being rich of lysine and arginine. New possibilities of examining the proteins in leukocytes in a cytochemical way as well as in blood- and bone-marrow cells are provided by colouring with palatine fast black, the use of which also points to certain differences in the chromatin of lymphocytes and neutrophils.     

Ultrastructural cytochemistry of atrial muscle cells

Summary Sections of atrial cardiocytes from young rats were subjected to radioautography after a single intravenous injection of L-leucine-4,5 ³H to identify the sites of synthesis and to follow the migration of newly-formed proteins. As early as 2 min after injection of L-leucine ³H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 5 min, most of the label had migrated from the RER to the Golgi complex. Some label was already present over specific granules by 2 min but the peak was reached at 1 h. By 4 h, the label over the specific granules had diminished, possibly indicating a release of newly-synthetized secretory material outside the cell. The label over myofilaments and Z-bands was relatively high at most time intervals, suggesting an early and important incorporation of leucine into the contractile and structural proteins of these organelles. The label over the cytosol was initially high and increased even further at 5 and 20 min but decreased to a very low level at 4 h. In contrast, the label over the cell surface rose continuously and peaked at 4 h. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the cytosol before reaching their destination.     

The cytochemistry of Limulus eggs.

Cytochemical studies on uninseminated mature eggs of Limulus demonstrate the presence of carbohydrates, lipids and proteins in the egg envelopes and yolk. The vitelline envelope, cortical region and yolk are rich in 1,2-glycols, with the vitelline envelope, containing fewer reactive 1,2-glycol groups than other components of the egg. Neutral mucopolysaccharides are found in the cortical region and yolk, but only the cortical region of the eggs demonstrate the presence of sulfated mucosubstances (which are in part glycoprotein in nature) and glucose-6-phosphatase. Protein is evident in all egg components. Biochemical analysis demonstrate the protein in the egg envelopes of uninseminated eggs is composed of sixteen amino acids while that of developing eggs contain seventeen amino acid residues. Electrovalent linkages and non-S-S- covalent linkages between protein chains are shown to be instrumental in maintaining the stuctural integrity of Limulus egg envelopes. Neutral lipids, unsaturated lipids, phospholipids and fatty acids are demonstrated in yolk bodies and lipoproteins, unsaturated lipids and fatty acids constitute part of the egg envelopes. DNA is concentrated in the cortical region and the yolk bodies     

Ultrastructural cytochemistry of human spermatozoa]

Enzymatic activities and repartition of glycoproteins were studied with electron microscopy in human ejaculated spermatozoa. Enzymatic activities are localised in the head of spermatozoon: arylsulfatase in the acrosome, acid phosphatase in the periacrosomal cytoplasm. Phosphotungstic acid at low pH and collo?dal iron allow detection of glycoproteins and acid groups on the sperm cell surface. Glycoproteins are present in the acrosome. These results are slightly different to those obtained in other species.     

Ultrastructural carbohydrate cytochemistry of gastric epithelium

Synopsis On examination with ultrastructural methods for visualizing thevicinal glycols and acid groups of complex carbohydrates, the most superficial surface epithelium of the rat gastric corpus displayed biphasic mucous droplets consisting of a cortex of hexose-rich (i.e. periodate-reactive) neutral mucosubstance and an uncharacterized denser core plus monophasic droplets with the neutral mucosubstance. In many surface epithelial cells of the foveolae, the biphasic and monophasic droplets with the neutral mucosubstance intermingled in varying proportions with monophasic droplets showing uniform periodate reactivity, a variable degree of dialyzed ironbinding—demonstrative of acidic glycoconjugate, and high iron—diamine affinity—demonstrative of sulphomucin. Deep foveolar epithelium displayed only monophasic droplets, most of which contained acidic periodate-reactive complex carbohydrate. Underiying cells, designated isthmus cells, exhibited monophasic or occasional biphasic granules containing sulphated, hexose-rich mucosubstance. Nascent droplets or granules near the Golgi zone differed from the mature organelles in the distribution of the glycoconjugate. Mucous neck cells occupied a deeper stratum and displayed a uniform population of monophasic mucous droplets with a loose meshwork of neutral mucosubstance.Techniques for demonstrating hexoses ultrastructurally stained all Golgi cisternae in the mucigenic epithelium, showing increasing reactivity toward the maturing face. Distinctive cistemae with moderate reactivity in the Golgi complex of isthmus cells were interpreted as GERL. Acidic mucosubstances were visualized only in the inner, mature cisternae of the Golgi complex of cells storing acidic glycoconjugates, and not in cisternae interpretable as GERL.The apical plasmalemma of isthmus cells uniquely exhibited abundant sulphated glycoconjugate and that of parietal cells revealed a less prominent, periodic neutral mucosubstance. Lateral and basal plasmalemmae varied from unstained to slightly reactive; basement membranes showed moderate reactivity with methods for visualizing complex carbohydrates. Abundance of glycogen further characterized surface epithelial cells of the corpus and of some parietal cells