Elias Meezan

A simple, versatile, nondisruptive method for the isolation of morphologically and chemically pure basement membranes from several tissues☆

Abstract A simple procedure has been developed for the isolation of ultrastructurally pure, intact basement membranes from bovine retinal and brain blood vessels, rabbit renal tubules and rat renal glomeruli. By this procedure, cell membranes and intracellular materials are selectively solubilized with 4% sodium deoxycholate to yield morphologically and chemically intact basement membrane preparations. Therefore, this method appears to be a versatile, nondisruptive procedure for the isolation and characterization of basement membranes from a variety of tissues. Its applicability has been demonstrated by the preparation for the first time of isolated basement membranes from non-renal mammalian blood vessels.

Isolated Brain Microvessels: A Purified, Metabolically Active Preparation from Bovine Cerebral Cortex

A purified, metabolically active preparation of brain microvessels was isolated from bovine cerebral cortex by using a simple procedure involving mild disruption of the tissue by homogenization and trapping of the vessels on nylon sieves. This preparation permits in vitro metabolic and structural studies of small blood vessels.

Ultrastructural and biochemical analyses of isolated basement membranes from kidney glomeruli and tubules and brain and retinal microvessels

We have developed a simple, versatile, and nondisruptive method for isolating basement membranes (BM) from several organ subfractions. In the past, investigators have employed sonication as an almost universal step in the isolation and purification of BM. This procedure, however, frequently results in cellular contamination and fragmentation of the starting material. Our method relies on the selective solubilization of cell membranes, intracellular protein, and plasma proteins with the detergents Triton X-100 and deoxycholate. This technique yields a preparation of BM which is ultrastructurally intact, nonfragmented, and which retains its histoarchitectural relationships. Basement membrane isolated from kidney glomeruli and tubules and brain and retinal microvessels show continuous sheets, free of cellular debris. Interstitial collagens are virtually excluded. The BM from different sources exhibit a wide variation in thickness and morphology emphasizing a nonunitary concept of BM. In all cases, however, isolated BM are ultrastructurally indistinguishable from their in vivo counterparts. Amino acid and carbohydrate analyses of previously well-characterized BM (lens capsule and glomerular BM) show that following isolation by detergents their chemical compositions compare favorably with reported values. Other sources of BM show similar compositions with high contents of glycine, hydroxyproline, and glutamic acid. All BM show nearly equimolar amounts of glucose and galactose while mannose, fucose, and hexosamines are present in smaller quantities. We conclude that our method of BM isolation may be of particular value where studies of both the fine structure and chemical composition of BM are to be examined.

pH gradient elution of human IgG1, IgG2 and IgG4 from protein A-Sepharose

Pooled human serum having a normal IgG subclass content was chromatographed on a column of protein A-Sepharose. The immunoglobulins that bound to the column at pH 7.0 were eluted with a pH gradient generated by 3 equal volumes of citrate/phosphate buffer at pH 5.0, 4.5 and 2.2. The elution pattern consisted of two major overlapping peaks centered 0.4 pH units apart in the pH gradient; on average the first peak centered at pH 4.7, and the second centered at pH 4.3. Upon second passage of each component, single major peaks centered at the appropriate pH were seen. The subclass distributions of the re-chromatographed peaks were as follows: the high pH-eluting IgG contained less than 1% IgG1, 95% IgG2 and 5% IgG4; the low pH-eluting IgG contained 90% IgG1, 6% Ig2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. Chromatography on protein A-Sepharose provides a means for separating normal human IgG1 from IgG2 and may therefore prove useful as an additional tool for studying the relative biological role of these IgG subclasses.

The pH-dependent binding of goat IgG1 and IgG2 to protein A-Sepharose

Abstract Hjelm et al. (1972) introduced protein A-Sepharose to separate protein A-reactive from nonreactive immunoglubulins. In the conventional use of this procedure, all bound immunoglobulins are eluted in a single step with 1 M acetic acid. We used instead a pH gradient to elute column-bound goat immunoglobulins and resolved two major IgG components; one eluting with a peak at pH 6.7 and the other at pH 5.8. We investigated the relationship between these components and the known IgG subclasses which, in the goat, are separable by DEAE-cellulose chromatography. Protein A-Sepharose chromatography of DEAE fractions indicates that the second DEAE peak, in which IgG1 is known to predominate, corresponds to the IgG eluting at pH 6.7 and that the first DEAE peak, in which IgG2 predominates, corresponds to the IgG eluting at pH 5.8. Commercial anti-bovine IgG1 reacted strongly upon double immunodiffusion with the pH 6.7 IgG and only slightly with the pH 5.8 IgG; anti-bovine IgG2, however, failed to react with either sub-population of goat IgG.

Metachromatic staining with Coomassie Brilliant Blue R-250 of the proline-rich calf thymus histone, H1

In this report, we show that calf thymus histone 1 stains metachromatically with Coomassie Brilliant Blue R-250. Histone 1 gel bands appear red instead of the more familiar blue color characteristic of the vast majority of proteins. The red color and the spectral properties of histone 1 bands are qualitatively similar to those of collagen and procollagen bands which, as previously shown by others, also stain metachromatically (Micko, S. and Schlaepfer, W.W. (1978) Anal. Biochem. 88, 566-572 and McCormick, P.J., Chandrasekhar, S. and Millis, A.J.T. (1979) Anal. Biochem. 97, 359-366). In contrast to histone 1, histones 2A, 3 and 4 stain blue; histone 2B also stains predominantly blue, but with a faint red tint. Both red and blue bands display an absorption maximum in the vicinity of 560 nm, but red bands display an additional maximum in the vicinity of 530 nm. There are quantitative differences between the red bands; the 530 nm peak is more prominent in the spectrum of the collagen band than in the spectrum of the histone 1 band. The spectra of the blue bands are all very similar except that the spectrum of the histone 2B band is shifted slightly toward lower wavelengths. To account for the spectral differences between protein bands, we propose a model in which closely-spaced proline residues are responsible for the chromotropic effect. Localized concentrations of proline residues are present in both collagens and histone 1 and a small cluster is also present in histone 2B.

Preparation and composition of alveolar extracellular matrix and incorporated basement membrane

A cell-free alveolar extracellular matrix has been prepared from the lungs of several species of young and adult animals by a procedure consisting of extraction of parenchymal fragments with the detergents Triton X-100 and sodium deoxycholate. The isolated extracellular matrix preparations were ultrastructurally heterogeneous consisting of amorphous basement membranes and associated interstitial collagen and microfibrillar components as shown by histochemical staining and their electron microscopic appearance. The amino acid and carbohydrate compositions of these preparations had a collagenous nature which resembled in many respects that of some ultrastructurally pure basement membranes. Urea extraction of extracellular matrix from adult animals solubilized a distinctly less collagenous fraction which was particularly rich in the acidic amino acids, aspartic and glutamic acids, and had a chemical similarity to acidic structural glycoproteins. Extraction of adult samples with ureamercaptoethanol and pepsin digestion of the insolubilized residues gave more collagenous fractions. Our ultrastructural and chemical examination of these alveolar extracellular matrix preparations clearly indicate that they cannot properly be designated as pure alveolar basement membranes. These findings and the anatomical characteristics of the alveolar blood-air interface make it probable that preparations isolated by others using sonication procedures, with chemical characteristics similar to those reported here, were not ultrastructurally pure alveolar basement membranes as reported but were actually heterogeneous mixtures of basement membranes, interstitial collagens and microfibrils. It would appear that the alveolar basement membrane cannot be isolated in a form comparable to ultrastructurally pure basement membranes such as that of the renal glomerulus.

A simple apparatus for the continuous monitoring of 14CO2 production from several small reaction mixtures

Abstract A simple apparatus is described which permits the continuous monitoring of 14 CO 2 production from ten separate reaction mixtures simultaneously. The device is relatively simple and inexpensive to construct, makes use of small disposable incubation vials, and allows complete trapping of all 14 CO 2 evolved in scintillation vials, where it can be easily counted. The use of this apparatus to determine the rates of metabolism by glomeruli of 14 C-labeled substrates to 14 CO 2 is described.

Rapid Isolation of Type II Pneumocytes with Magnetic Removal of Macrophages

A method is described for the rapid preparation of lung cell fractions enriched in type II alveolar pneumocytes. Isolated perfused rabbit lungs are exposed to Fe3O4 by tracheal lavage, which permits pulmonary alveolar macrophages to phagocytize the particles. Alveolar epithelial cells are then selectively freed from the basement membrane matrix by critical placement of collagenase and elastase. Detached cells are harvested either by repeated tracheal lavage or by mincing the lobes and filtering freed cells through a series of nylon mesh sieves. Iron oxide-containing macrophages are then removed from the harvested cells by a strong magnetic field. A final sizing of the macrophage-depleted suspension yields a preparation enriched in alveolar type II cells. Eight million viable cells (95% type II) were obtained per rabbit lung when harvested by lavage, while 32 x 10(6) (88% type II) cells were obtained from minced lungs. These values for cell yield and relative purity are comparable to previously described separation methods that depend upon differences in cell density or size. A major advantage of the magnetic separation procedure is the substantially shortened preparation time, typically 2 hr instead of 4. The viability (90-95%), oxygen consumption (88 nmol/10(6) cells/hr), and incorporation of [14C]acetate and [14C]choline (0.44 and 0.115 nmol/10(6) cells/hr, respectively) indicate that these cells will be suitable for pharmacologic and toxicologic investigation.

Basement membrane biosynthesis by isolated bovine retinal vessels: Incorporation of precursors into extracellular matrix

Abstract The biosynthesis of microvascular basement membrane has been demonstrated in a purified preparation of isolated bovine retinal vessels. With the use of a detergent extraction procedure to isolate morphologically intact retinal vessel basement membrane, the incorporation of labeled amino acids and carbohydrates into the purified extracellular matrix was shown. Electron microscopy demonstrated the purity of the microvessel preparations and the basement membrane derived from them, confirming that the incorporation observed represented incorporation of radioactive precursors into ultrastructurally intact microvascular basement membrane. The incorporation of amino acids and carbohydrates into the basement membrane was sensitive to heat inactivation, respiratory poisons, and inhibitors of protein and collagen synthesis. Only a small fraction of the proline label deposited in the extracellular matrix was digested by collagenase treatment, in agreement with the small amount of labeled hydroxyproline found in the isolated basement membrane, indicating that while some synthesis of collagen is observed, the bulk of incorporation occurring in this system appears to be into noncollagenous glycoprotein components of the basement membrane. The results are in agreement with different rates of secretion and deposition of glycoprotein and collagen components into the basement membrane matrix. This is the first demonstration of basement membrane biosynthesis by the isolated retinal microvasculature and represents a promising system for the study of the metabolic basis for the marked pathology of the vessel wall occurring in diseases such as diabetes mellitus.