Sensitivity of a Lamb wave sensor with 2 microm AlN membrane.

Anti-symmetrical Lamb wave mode A0 presents a large sensitivity to mass loading and can be used in contact with liquids with a small attenuation. The advantages of this system are the possibility to get a large mass sensitivity. The sensitivity increases when the thickness of membrane decreases. Therefore the problem is to obtain thin piezoelectric membranes. A membrane of AlN with a thickness of 2 microm has been made. The measured mass sensitivity with a fluid is 200 cm(2) g(-1). In a practical use point of view, the problem in this kind of sensor is its temperature sensitivity. In order to reduce effective temperature sensitivity, a device with thin metallic strips is presented. On the same membrane two different waves with perpendicular propagating directions are produced. Experimentally, temperature sensitivity is rather different depending on the propagation direction but mass sensitivity is almost the same, this allows distinguishing temperature effects from those due to mass loading on the frequency shift measurements.

An albumin-coated polyester arterial graft: in vivo assessment of biocompatibility and healing characteristics.

The albumin-coated vascular graft (ACG) and its uncoated polyester substrate, the Vascular II (V-II), were evaluated in terms of biocompatibility and biofunctionality using two in vivo animal studies. Biocompatibility and immunoreactivity were assessed by implanting intraperitoneally in the rat small segments of the ACG and the V-II graft and harvesting them with their surrounding tissue 3d, 1, 2 and 4 weeks later. Cytofluorometric determination of total T cells (CD3), the ratio of CD4/CD8 subsets and the percentage of IL-2 receptor-positive T cells in the peripheral blood has revealed that no significant difference in any of the T cell populations was found between the ACG and the V-II graft. The cellular reactivity of the ACG in terms of acid phosphatase activity at the implant side was significantly greater at 3 d but not at longer periods. Biofunctionality was evaluated by implanting both grafts as a thoracoabdominal vascular bypass in dogs for 11 different periods ranging from 4 h to 6 months. The rate of albumin resorption was such that traces were still present at 1 month, but no longer observable at 2 months. Tissue incorporation into the graft wall was earlier for the V-II (2 weeks) than for the ACG (4 weeks), which showed complete encapsulation, tissue incorporation and endothelialization after 2 months in vivo. Only small differences were observed between both grafts in terms of platelet and fibrin uptake on the luminal surface. The prostacyclin/thromboxane A2 ratio increased to a level higher that 1.0 aorta within 1 month for the V-II and 4 months for the ACG. In conclusion, the Bard ACG has demonstrated excellent biocompatibility in terms of blood T cell behaviour and acid phosphatase activity at the implant site. Finally, its healing response is equivalent to that of the uncoated Dacron prosthesis once the albumin coating has been resorbed.

Albumin impregnated vascular grafts: albumin resorption and tissue reactions.

This study aimed to determine the kinetics of albumin resorption from and the healing of two types of albumin impregnated Vasculour II (Bard Cardiovascular) Dacron grafts (ACG-A and ACG-B) using whole blood preclotted Vasculour II Dacron grafts (without albumin) as controls (PCC). Prostheses measuring 4 mm ID x 50 mm length were implanted in the aortoiliac position in 24 dogs (ACG-A n = 12, ACG-B n = 24, PCC n = 12) and explanted after 1, 2 4, and 6 months. Platelet count, platelet aggregometry to 10(-5) M ADP, prothrombin time (PT), and partial thromboplastin time (PTT) were determined preoperatively and at explantation. Sections of the explanted grafts were assayed for human albumin by immunohistochemical techniques utilizing a rabbit polyclonal mono-specific antibody for human albumin followed by the addition of a biotinylated goat anti-rabbit IgG. Immunoperoxidase staining was then performed using Avidin D horse-radish peroxidase. Histology of the grafts (light microscopy, scanning electron microscopy, and transmission electron microscopy) as well as percent thrombus free surface area (TFSA) by computerized planimetry were also determined. Seven of 48 grafts were occluded (85.4% patency) with no difference among the three groups. Platelet aggregometry was not predictive of graft patency. No change in PT or PTT occurred nor was there any difference among the three groups. Retained albumin was detected in every one-month explant but not beyond that time, with the sensitivity for detecting human albumin in this assay being 20 mg albumin per gram of Dacron. All ACG explants at one month revealed inner capsular fibrin coagula not present in PCC specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Acellular vascular matrix: a natural endothelial cell substrate.

A preliminary assessment was made of the acellular vascular matrix graft as a substrate for endothelial cell seeding, with respect to surface pretreatment (none versus fibronectin and/or serum) and presence of exogenous growth factor. Arteries were harvested from greyhounds and exposed to a sequential detergent extraction process to produce the acellular vascular matrix. Human umbilical vein endothelial cells were grown in tissue culture, harvested in first passage, then seeded at 10(5) cells/cm2 on sections of acellular vascular matrix and on gel-coated polystyrene positive controls. After 18 hour incubation, endothelial cell-seeded acellular matrices were fixed and processed for histologic and planimetric analysis; control wells were fixed and endothelial cells were counted by planimetry. Pretreatment of the acellular vascular matrix was found to have no effect on the percentage of endothelial cell coverage of the matrix. There was significantly better endothelial cell coverage of the acellular matrix than on matched gel-treated polystyrene control wells. Withdrawal of growth factor resulted in a significant reduction in endothelial cell coverage for all acellular vascular matrix groups. Growth factor withdrawal also significantly reduced attachment of endothelial cells on gel-treated polystyrene. Cell surface area was significantly smaller when growth factor was withdrawn from all groups except from the acellular vascular matrix without pretreatment. We conclude that: (1) the acellular vascular matrix is conductive to endothelial cell adherence and spreading even without pretreatment; and (2) sudden withdrawal of exogenous growth factor may impair early coverage of substrates by endothelial cells due to an effect on their adherence or spreading.

Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates.

The use of fluorescein-avidin or rhodamine-avidin conjugates in conjunction with biotinylated secondary antibodies for indirect immunohistology frequently results in pronounced nonspecific nuclear staining in kidney sections. This nonspecific nuclear staining can be effectively blocked by using 5% (w/v) nonfat dry milk in buffered saline as the diluent for the avidin conjugates. In contrast, 5% (w/v) bovine serum albumin, a commonly used blocking agent, has only a modest effect. Nonfat dry milk is also effective as a blocking agent and carrier when used in fluorescence-activated cell sorter (FACS) analysis. These results emphasize the broad usefulness of milk-based blocking reagents.

The formation and biotransformation of cysteine conjugates of halogenated ethylenes by rabbit renal tubules.

The nephrotoxicity of chlorotrifluoroethylene ( CTFE ) was examined using isolated rabbit renal tubules suspensions. Exposure of the tubules to CTFE resulted in consumption of CTFE , formation of a glutathione conjugate and inhibition of active organic acid transport. Synthetic cysteine, N-acetylcysteine or glutathione conjugates of CTFE inhibited transport indicating S-conjugation as a possible toxic pathway. 1,2-dichlorovinyl glutathione ( DCVG ), a model synthetic glutathione conjugate, was used to examine the degradation and toxicity of these conjugates. DCVG inhibited rabbit renal tubule transport in vivo and in vitro. The DCVG was found to be degraded with the evolution of glutamine and glycine to produce the ultimate nephro-toxicant, dichlorovinyl cysteine. Dichlorovinyl cysteine is then bioactivated with the release of ammonia. This sequential degradation explains the latency of DCVG -induced renal transport inhibition relative to dichlorovinyl cysteine. It is now evident that certain halogenated ethylenes are capable of being biotransformed to glutathione conjugates in the kidney with their subsequent hydrolysis to nephrotoxic cysteine conjugates.

Holocene age of the Yuha burial: direct radiocarbon determinations by accelerator mass spectrometry.

The view that human populations may not have arrived in the Western Hemisphere before about 12,000 radiocarbon yr BP has been challenged by claims of much greater antiquity for a small number of archaeological sites and human skeleton samples. One such site is the Homo sapiens sapiens cairn burial excavated in 1971 from the Yuha desert, Imperial County, California. Radiocarbon analysis of caliche coating one of the bones of the skeleton yielded a radiocarbon age of 21,500 +/- 1,000 yr BP, while radiocarbon and uranium series analyses of caliche coating a cairn boulder yielded ages of 22,125 +/- 400 and 19,000 +/- 3,000 yr BP, respectively. The late Pleistocene age assignment to the Yuha burial has been challenged by comparing the cultural context of the burial with other cairn burials in the same region, on the basis of the site's geomorphological context and from radiocarbon analyses of soil caliches. In rebuttal, arguments in defence of the original age assignment have been presented as well as an amino acid recemization analysis on the Yuha skeleton indicating an age of 23,600 +/- 2,600 yr BP. The tandem accelerator mass spectrometer at the University of Arizona has now been used to measure the ratio of 14C/13C in several organic and inorganic fractions of post-cranial bone from the Yuha H. sapiens sapiens skeleton. Isotope ratios from six chemical fractions all yielded radiocarbon ages for the skeleton of less than 4,000 yr BP. These results indicate that the Yuha skeleton is of Holocene age, in agreement with the cultural context of the burial, and in disagreement with the previously assigned Pleistocene age of 19,000-23,000 yr.

Differentiation of the cardiac and pulmonary toxicity of monocrotaline, a pyrrolizidine alkaloid.

Monocrotaline, given to rats as a 20 mg/l solution in drinking water for 3 weeks, doubled the mass of the right heart and lung. The rise in lung mass preceded that of the heart. These increases were accompanied by increases in the absolute protein content of the two organs, together with increases in the rates of both protein and RNA syntheses. The increase in lung mass was not accompanied by a change in total collagen content, as measured by two independent methods: 4-hydroxyproline content and detergent fractionation. In contrast, the right ventricle showed more than a 4-fold increase in total collagen content. Total pulmonary lipids increased by 86%, but the lipid: protein ratio was unchanged. Right ventricular lipids were unchanged in amount but the lipid: protein ratio fell by 29%. Lung DNA:RNA ratio decreased 49% and right ventricle DNA:RNA ratio decreased 69%, indicating that both of these organs were responding to monocrotaline with hypertrophy. These results suggest that the processes of hypertrophy differ in the two organs: in the lung, there was no fibrosis despite a marked increase in dry weight, while right ventricular hypertrophy was characterized by increased collagen deposition. There was no alteration in the left ventricle in any of the parameters investigated.

Detergent-extracted small-diameter vascular prostheses.

The exact mechanism that leads to thrombosis of small-diameter vascular prostheses (less than 4 mm X greater than 6 cm) is unknown. This report presents preliminary patency and healing data on a sequential detergent-extraction technique for the preparation of autogenous small-diameter microvascular grafts. Fifteen carotid interpositional allografts (3 to 4 mm X 4 to 6 cm) were implanted in 15 mixed species adult greyhounds. Ninety days after implantation grafts were perfusion-fixed in situ, harvested, and evaluated by light microscopy and scanning and transmission electron microscopy. Two categories of acellular vascular matrix grafts were evaluated: non-cross-linked and cross-linked (1% carbodiimide). By objective morphologic analysis with blind random view, histologic sections were rated from 0 to 4 in five categories believed to be important for graft healing and patency. Overall graft patency was 80% (12 of 15), and there was no significant difference between cross-linked and non-cross-linked grafts. Non-cross-linked grafts were superior to cross-linked grafts in all areas of histologic evaluation except immunogenicity (p less than 0.01). Most important, non-cross-linked grafts demonstrated complete endothelial coverage (p less than 0.001). There was no significant difference (that is, normal) between control autografts and non-cross-linked grafts; however, there was a significant difference between control autografts and cross-linked grafts in all parameters except immunogenic reaction (p less than 0.01).

Cockroach larval-specific protein, a tyrosine-rich serum protein.

Larval-specific protein (LSP) is the most abundant protein in the hemolymph of cockroaches shortly before molting, but is rapidly cleared from the hemolymph during the molt (Kunkel, J. G., and Lawler, D. M. (1974) Comp. Biochem. Physiol. 47B, 697-710). Blatta orientalis LSP was purified by sedimentation in preparative sucrose gradients followed by 2-hydroxypropylamino-cellulose anion-exchange chromatography and gel filtration on a column of Bio-Gel A-1.5m. The amino acid composition of LSP includes 16.3 mol % tyrosine and 4.9 mol % phenylalanine, but virtually no cysteine and little methionine. The following physical properties were determined for LSP: R8 = 68.3 A, 8(20),w = 17.8, and V = 0.723. From these values an Mr = 507,900 was calculated. In electron micrographs, LSP appears as rectangular particles of 121 by 134 A. In disc polyacrylamide gel electrophoresis, native LSP exhibits a single band, but in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LSP is resolved into a doublet of closely spaced bands of Mr = 88,100 and 84,400 present in a ratio of 1.38:1. These data indicate that native B. orientalis LSP is a hexamer of subunits averaging approximately Mr = 86,000. Crossed immunoelectrophoresis of Blattella germanica larval serum indicates that LSP in that species is a hexamer composed of a random assortment of two subunits of different charge in the ratio 1.25:1. The amino acid composition and physical properties of LSP suggest that LSP may be the hemimetabolous analogue of the tyrosine- and phenylalanine-rich storage proteins of holometabolous insects.

Differential staining of collagens and non-collagens with Coomassie Brilliant Blue G and R.

The following proteins were subjected to electrophoresis in SDS gels and stained with both Coomassie Brilliant Blue R and Coomassie Brilliant Blue G: the pepsin-treated collagen types I, II, III and V, and non-pepsin-treated type IV collagen, and the non-collagens, laminin, fibronectin, myosin, beta-galactosidase, fibrin, phosphorylase b and serum albumin. The Coomassie Brilliant Blue G stain was formulated as in the dye-binding protein assay reagent of Bradford (Anal. Biochem. 72: 248-254, 1976). Coomassie Brilliant Blue R prominently stained all polypeptides, but the collagen chains, including the type IV chains, stained metachromatically (red or pink) while the non-collagens stained orthochromatically (blue-violet). In the Bradford reagent, however, only the non-collagens and the intact type IV chains were prominently stained; the pepsin-treated collagen chains were virtually undetectable provided that detergent had been exhaustively removed prior to immersion in the stain. Metachromatic staining with Coomassie Brilliant Blue R is attributed to the presence of closely-spaced proline and hydroxyproline residues in sequences from triple-helical domains. The staining of type IV chains with the Bradford reagent is attributed to the presence of binding sites in the sequences from the non-triple-helical domains only, since such binding sites are absent from chains derived from the pepsin-treated collagens.

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.

Selective solubilization of two populations of polypeptides from bovine retinal basement membranes.

Basement membranes isolated from the entire neural retinae of normal cows consist of a heterogeneous mixture of polypeptides resolvable as two major populations, one collagenous and the other non-collagenous. The non-collagenous population consists of disulfide-bonded aggregates of which the major fraction is soluble in lithium dodecyl sulfate (LDS) at 4 degrees C without reducing agent, with the remainder requiring reduction for solubilization at 4 degrees C. Only the second 4 degrees C extract is contaminated with small amounts of hydroxylated amino acids. The non-collagenous population consists of up to 25 polypeptides detectable in sodium dodecyl sulfate (SDS) gels, all of which stain blue with Coomassie Blue R-250, are insensitive to bacterial collagenase and migrate on or slightly below the central diagonal in urea-SDS/SDS electrophoresis. The collagenous population is soluble in LDS at 100 degrees C under reducing conditions and has a collagenous amino acid composition, although consisting of only 272 glycine residues/1000. This fraction consists of four major and four minor polypeptides detectable in SDS gels, all of which stain red (metachromatically) with Coomassie Blue R-250, are degraded by bacterial collagenase and migrate below the central diagonal in urea-SDS/SDS electrophoresis. The four major collagenous polypeptides are larger than the alpha-chains of type I collagen. In total, the solubilized proteins account for 60% of the general protein, but only 30% of the sum of hydroxyproline and hydroxylysine. The insoluble residue has a collagenous amino acid composition similar to that of the 100 degrees C extract, but with lower 3-hydroxyproline and hydroxylysine contents.

Coomassie Blue R-250 metachromatic staining of histone 5 from goose and chicken erythrocytes.

1. Histone 5 stains metachromatically with Coomassie Blue R-250, a property shared only with collagens and procollagens, histones 1 and 2B, and possibly certain proline-rich salivary gland proteins. 2. Coomassie Blue-stained goose and chicken H5 induce the same degree of metachromasia, which is intermediate between that induced by H1 and H2b. 3. The absorption spectra of all Coomassie Blue-stained gel bands consist of a primary maximum at 555 nm, but the absorption spectra of H5 gel bands also include a prominent shoulder in the vicinity of 525 nm, a region in which H1 gel bands display a secondary maximum. 4. The possible role of proline-rich sequences in the induction of metachromasia is discussed.

Isolation of proline and hydroxyproline from fossil bone.

We describe a simple and rapid procedure for the isolation of proline and hydroxyproline from fossil bone based on the isolation of gelatin, hydrolysis, purification with XAD-2, deamination of primary amino acids with aqua regia, and separation of the imino acids by cation exchange chromatography. This procedure will provide material for accurate bone carbon dating, and stable carbon isotope ratio determinations for the evaluation of paleodiets.

Preparation of cell-free extracellular matrix from human peripheral nerve.

The extracellular matrix of human peripheral nerve, which is mainly basement membrane and fibrillar collagen, has been prepared by a procedure involving extensive detergent extraction of isolated endoneurium and perineurium obtained from various nerves. The ultrastructure of the isolated nerve extracellular matrix was indistinguishable from that seen in sections of intact nerve, indicating that the extraction procedure preserved the morphological integrity of these connective tissue components. The amino acid and carbohydrate compositions of the nerve extracellular matrix preparations were typically collagenous in nature containing a high content of glycine, proline, 4-hydroxyproline, and alanine and significant amounts of lysine and hydroxylysine. The preparations contained virtually no 3-hydroxyproline and low content of glucose and galactose compared to pure basement membranes, indicating that interstitial rather than basement membrane collagens predominated. This preparation appears well-suited to both the ultrastructural and biochemical study of the extracellular matrix of peripheral nerve.

The addition of SDS to the Bradford dye-binding protein assay, a modification with increased sensitivity to collagen.

The standard Bradford protein assay is insensitive to collagen. But if a small, sub-threshold amount of SDS is added to the sample, the response to collagen is increased by at least an order of magnitude, while, on average, the sensitivity for non-collagens is decreased by approximately a factor of 2. As a result, comparable color formation is achieved with both collagen and non-collagens. The addition of protein to a sub-threshold amount of SDS results in the formation of a green color measurable as an increase in absorbance at 700 nm, in contrast to the blue color measured at 595 nm in the standard assay. Depending upon the source, the threshold level for SDS varies from 30 to 50 microgram. The response to protein is linear up to approximately 40 microgram of protein per ml of reagent.