Synthesis of bone-like micro-porous calcium phosphate/iota-carrageenan composites by gel diffusion

Brushite and octacalcium phosphate (OCP) crystals are well-known precursors of hydroxylapatite (HAp), the main mineral found in bone. In this report, we present a new method for biomimicking brushite and OCP using single and double diffusion techniques. Brushite and OCP crystals were grown in an iota-carrageenan gel. The aggregates were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), infrared spectroscopy (IR) and thermal gravimetric analysis (TGA). SEM revealed different morphologies of brushite crystals from highly porous aggregates to plate-shaped forms. OCP crystals grown in iota-carrageenan showed a porous spherical shape different from brushite growth forms. The XRD method demonstrated that the single-diffusion method favors the formation of monoclinic brushite. In contrast, the double diffusion method was found to promote the formation of the triclinic octacalcium phosphate OCP phase. By combining the different parameters for crystal growth in carrageenan, such as ion concentration, gel pH and gel density, it is possible to modify the morphology of composite crystals, change the phase of calcium phosphate and modulate the amount of carrageenan inclusion in crystals. This study suggests that iota-carrageenan is a high-molecular-weight polysaccharide that is potentially applicable for controlling calcium phosphate crystallization.

Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.

Evidence for the involvement of proximal chromosome 3P loci in the progression of Von Hippel-Lindau related tumors

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of retinal and central nervous system hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic islet cell tumors (PICT). The VHL gene maps to chromosome 3p25 and has been shown to be mutated in 57% of sporadic cases of RCC, implicating VHL in the genesis of RCC. We report a multigeneration VHL kindred in which four affected female siblings developed PICT at early ages. Analysis of the three coding exons of the VHL gene in this family revealed a single, missense mutation in codon 238. Inheritance of the 238 mutation has been reported to correlate with a 62% risk of pheochromocytoma development. In this kindred, all affected individuals carried the mutation as well as one additional sibling who showed no evidence of disease. Clinical screening of this individual indicated small ($<$1 cm) pancreatic and kidney tumors. Results suggest that inheritance of the codon 238 mutation does not correlate with early onset pheochromocytoma. Rather, the only individual in the pedigree with pheochromocytoma was the proband's mother who developed bilateral pheochromocytoma at the age of 62. Thus, the VHL codon 238 mutation may predispose to late onset pheochromocytoma in this family; however, it does not explain the preponderance of PICT in the third generation since this mutation has not been reported to increase the risk of developing pancreatic lesions. This suggests that inheritance of the codon 238 mutation and subsequent somatic inactivation of the wild type allele of the VHL gene may not be sufficient to explain the initiation and subsequent progression to malignancy in VHL-associated neoplasms. Since the two tumor types that most frequently progress to malignancy are RCC and PICT, we asked whether loss of heterozygosity (LOH) could be detected proximal to the VHL gene on chromosome 3 in distinct regions of 3p previously implicated by LOH and cytogenetic studies to contain tumor suppressor loci for RCC. LOH was performed on high molecular weight DNA isolated from peripheral blood and frozen tumor tissue of family members using microsatellite markers spanning 3p. Results indicated LOH for all informative 3p loci in tumor tissue from affected individuals with PICT. LOH was detected along the entire length of the chromosome arm and included the proximal region of 3p13-14.2 implicated in the hereditary form of renal cell carcinoma.^ If 3p LOH were a critical event in pancreatic islet cell tumorigenesis, then it should be expected that LOH in sporadic islet cell tumors would also be observed. We expanded LOH studies to include sporadic cases of PICT. Consistent LOH was observed on 3p with a highest frequency LOH in the region 3p21.2. This is the first evidence for an association between chromosome 3 loci and pancreatic islet cell tumorigenesis. (Abstract shortened by UMI.) ^

AN ANALYSIS OF THE DNA DAMAGE INDUCED BY NICKEL COMPOUNDS IN CHINESE HAMSTER OVARY CELLS (CARCINOGENESIS, HETEROCHROMATIN, CYTOTOXICITY, REPLICATION, PROTEIN CROSSLINKING)

The carcinogenic activity of water-insoluble crystalline nickel sulfide requires phagocytosis and lysosome-mediated intracellular dissolution of the particles to yield Ni('2+). This study investigated the extent and nature of the DNA damage in Chinese hamster ovary cells treated with various nickel compounds using the technique of alkaline elution. Crystalline NiS and water-soluble NiCl(,2) induced single strand breaks that were repaired quickly and DNA-protein crosslinks that persisted up to 24 hr after exposure to nickel. The induction of single strand breaks was concentration dependent at both noncytotoxic and lethal amounts of nickel. The induction of DNA-protein crosslinks was concentration dependent but was absent at lethal amounts of nickel. The cytoplasmic and nuclear uptake of nickel was concentration dependent even at the toxic level of nickel. However, the induction of DNA-protein crosslinks by nickel required active cell cycling and occurred predominantly in mid-late S phase of the cell cycle, suggesting that the lethal amounts of nickel inhibited DNA-protein crosslinking by inhibiting active cell cycling. Since the DNA-protein crosslinking induced by nickel was resistant to DNA repair, the nature of this lesion was investigated using various methods of DNA isolation and chromatin fractionation in combination with SDS-polyacrylamide gel electrophoresis. High molecular weight, non-histone chromosomal proteins and possibly histone 1 were preferentially crosslinked to DNA by nickel. The crosslinked proteins were concentrated in a magnesium-insoluble fraction of sonicated chromatin (5% of the total) that was similar to heterochromatin in solubility and protein composition. Alterations in DNA structure and function, brought about by the effect of nickel on protein-DNA interactions, may be related to the carcinogenicity of nickel compounds. ^

MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

THE LONG-TERM GROWTH OF NORMAL HUMAN B CELLS

The feasibility of establishment of continuously proliferating growth factor-dependent human B lymphocytes was investigated. Normal B lymphocytes prepared from peripheral venous blood were stimulated with a variety of known polyclonal B cell activators, in the continuous presence of various cytokine preparations. Continuously proliferating growth factor-dependent B cell populations were obtained from cultures activated with either insoluble anti-IgM ((mu)-chain specific), soluble anti-IgM, heat-killed Staphylococcus aureus Cowen I (SAC), or dextran sulphate (DxS), in the continuous presence of exogenously added growth factor preparations containing either IL-1, IL-2 and BCGF, or BCGF alone. Although growth factor-dependent B cell lines were obtained via all three methods of activation, the correlation of mode of activation and growth factor preparation proved to be critical. B cell lines could not be established with anti-(mu) activation in the presence of only BCGF; however, B cell lines were successfully obtained with SAC or DxS activation from those cultures continuously replenished with only BCGF. These cultured B lymphocyte populations were routinely maintained in logarithmic-phase growth in the presence of exogenously added growth factor, and exhibited a population doubling time of approximately 36 hours. They were shown to specifically absorb BCGF, suggesting the presence of membrane receptors for it. Also, these cultured B cells have been utilized for the development of a microassay for the assessment of a M(,r) 12,000-14,000 B cell growth factor activity that is accurate, sensitive, and precise. The pronounced sensitivity of this bioassay beyond that of the conventional peripheral blood B cell assay has aided in the purification to homogeneity of natural product extracellular BCGF (EC-BCGF), and in the determination of the nucleotide sequence for a gene coding for a protein exhibiting BCGF activity. Additionally, these B cell lines specifically absorb, and proliferate in the presence of, an affinity-purified M(,r) 60,000 trypsin-sensitive intracellular protein derived from freshly isolated human T lymphocytes, providing evidence for a putative intracellular precursor of EC-BCGF, or a novel high molecular weight BCGF species. ^

Physiologic and pathophysiologic fundamentals of proteinuria a review

The term proteinuria is taken to mean abnormally high protein excretion in the urine. Proteinuria is the consequence of glomerular filtration of plasma proteins, their subsequent reabsorption by the proximal tubular cells and secretion of protein by the tubular cells and distal urinary tract. In physiological conditions, the structural integry of the glomerular filtration barrier prevents the abnormal passage of albumin (molecular mass 66 kDa) and high-molecular-weight proteins (> 66 kDa), whereas the passage of low-molecular-weight proteins (< 66 kDa) is almost completely unrestricted. Proteins that arrive the tubular lumen are reabsorbed by endocytosis after binding to the megalin-cubilin complex. An increased load of proteins in the tubular lumen leads to the saturation of the reabsorptive mechanism and higher urinary protein excretion. Proteinuria can originate from prerenal, renal and postrenal causes. Elevated tubular protein concentrations have been recognized to be toxic to tubular cells and associated with the progression of chronic renal disease. Therefore, the quantitative and qualitative evaluation of proteinuria is important for the diagnosis of renal disease.

TimX, a novel player in protein import across the inner mitochondrial membrane of T. brucei

Mitochondrial protein import is an essential function of the unique mitochondrion in T. brucei as roughly 1000 different nuclear encoded proteins need to be correctly localized to their mitochondrial subcompartment. For this reason the responsible import machinery is expected to be similarly complex as in other Eukaryotes. This was recently demonstrated for the translocation machinery in the outer mitochondrial membrane. In contrast, the composition of the inner membrane import machinery and the exact molecular pathway(s) taken by various substrates are still ill-defined. To elucidate this further, we performed a pulldown analysis of epitope tagged TbTim17 in combination with quantitative mass spectrometry. By this we identified novel components of the mitochondrial import machinery in trypanosomes. One of these, TimX, is an essential mitochondrial membrane protein of 42 kDa that is unique to kinetoplastids. This protein migrates on Blue Native PAGE in a high molecular weight complex similar to TbTim17. Ablation of either of the two proteins leads to a destabilization of the complex containing the other protein. Furthermore, its involvement in protein import could be demonstrated by in vivo and in vitro protein import assays. This corroborates that TimX together with TbTim17 forms a protein import complex in the inner mitochondrial membrane. As TbTim17 the TimX protein was subjected to pulldown analysis in combination with quantitative mass spectrometry. The overlap of candidates defined by these two sets of IPs likely defines further components of the inner membrane translocase which are presently being analyzed. In summary our study on novel components of the trypanosome mitochondrial protein import system gives us fascinating new insights into evolution of the mitochondrion.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Their protein import systems are of similar complexity yet different composition than those in other eukaryotes. To elucidate the composition of the trypanosomal translocase of the inner mitochondrial membrane (TIM) we performed CoIPs of epitope-tagged TbTim17 and two other candidates in combination with SILAC-based quantitative mass spectrometry. This led to the identification of ten candidates for core TIM subunits. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a trypanosomatid-specific 42 kDa protein (Tim42) and two putative orthologues of inactive rhomboid proteases were analyzed further. All three proteins are essential in both life cycle stages and their ablation results in a strong protein import defect in vivo and in vitro. Blue native PAGE revealed their presence in a high molecular weight complex. Unlike anticipated, trypanosomes have a highly complex TIM translocase that has extensively been redesigned. None of the three novel TIM subunits has ever been associated with mitochondrial protein import. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. This suggests an exciting analogy between protein translocases of mitochondria and the ER.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is often considered as one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Its protein import systems are therefore of great interest. Recently, it was shown that the outer mitochondrial membrane protein translocase is of similar complexity yet different composition than in other eukaryotes (1). In the inner membrane however, only a single orthologue of the pore forming Tim17/22/23 protein family was identified and termed TbTim17. Based on this finding it has been suggested that, instead of separate TIM22 and TIM23 complexes as in other eukaryotes, trypanosomes may have a single multifunctional translocase of the inner mitochondrial membrane (TIM) of reduced complexity. To elucidate the composition of the trypanosomal TIM complex we performed co-immunoprecipitations (CoIP) of epitope-tagged TbTim17 in combination with SILAC-based quantitative mass spectrometry. This led to the identification of 22 highly enriched TbTim17-interacting proteins. We tagged two of the top-scoring proteins for reciprocal CoIP analyses and recovered a set of ten proteins that are highly enriched in all three CoIPs. These proteins are excellent candidates for core subunits of the trypanosomal TIM complex. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a novel trypanosomatid-specific 42 kDa protein, termed Tim42, and two putative orthologues of probably inactive rhomboid proteases were chosen for further analysis. All three proteins are essential in both life cycle stages and in a cell line that can grow in the absence of mitochondrial DNA. Additionally, their ablation by RNAi results in a strong protein import defect both in vivo and in vitro. Blue native PAGE reveals that Tim42, like TbTim17 is present in a high molecular weight complex. Moreover, ablation of either Tim42 or TbTim17 leads to a destabilization of the complex containing the other protein, suggesting a tight interaction of the two proteins. In summary our study shows that unlike anticipated trypanosomes have a highly complex TIM translocase that has extensively been redesigned. We have characterized three novel TIM subunits that have never been associated with mitochondrial protein import before. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. Our study provides insight into mitochondrial evolution over large phylogenetic distances and suggests an exciting analogy between protein translocation systems of mitochondria and the ER.

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight {HSPs} families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the {HSP60} family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.

STUDIES ON THE BIOLOGICAL ACTIVITY OF MACROMOLECULES MICROINJECTED INTO MAMMALIAN SOMATIC CELLS

Red Blood cell mediated and glass needle mediated microinjection technology was used to introduce macromolecules into mammalian somatic cells. The biological activities of DNA synthesis inducing factor(s) (Chapter 1), mitotic factor(s) (Chapter 2), and DNA coding for ovalbumin and thymidine kinase (Chapter 3) were studied following injection into mammalian somatic cells.^ Chapter 1. A cell undergoing DNA replication (S phase) contains a factor(s) that induces DNA synthesis prematurely in a G(,1) nucleus when an S phase cell is fused to a G(,1) cell. An assay for the active factor(s) was developed in which a mixture of s phase extract loaded red blood cells (RBC) and synchronous G(,1) HeLa cells was centrifuged onto Concanavalin A (Con A) treated coverslips and fused by PEG. This technique is called "Centrifusion". The synchronous G(,1) HeLa cells injected with S phase extract initiated DNA synthesis earlier than the control G(,1) cells mock injected with RBC loaded with buffer.^ Chapter 2. It has been demonstrated that fusion between a mitotic and an interphase cell usually leads to breakdown of the interphase nucleus, followed by condensation of the interphase chromatin into discrete chromosomes, a process termed premature chromosome condensation. I wanted to develop an assay for the mitotic factor(s) that induces premature chromosome condensation. Experiments were performed utilizing glass needle mediated microinjection of HeLa cell mitotic extract into interphase somatic mammalian cells in an attempt to induce premature chromosome condensation. However, I was not able to induce premature chromosome condensation in the interphase cells, probably because of an inability to introduce sufficient mitotic factor(s) into the cells.^ Chapter 3. A recombinant plasmid containing the chicken ovalbumin gene and three copies of the Herpes thymidine Kinase gene (pOV12-TK) was introduced into mouse LMTK('-) cell nuclei using glass needle mediated gene transfer resulting in LMTK('+) clones that were selected for in HAT medium. Restriction enzyme analysis of the high molecular weight DNA from 6 HAT medium survivor cell clones revealed the presence of one or at best only a few copies of the 12kb ovalbumin gene per mouse genome. Further analysis showed the ovalbumin DNA was not rearranged and was associated with high molecular weight mouse cell DNA. Each of the analyzed cell clones produced ovalbumin demonstrating that the biological activity of the microinjected ovalbumin was retained. ^

CHARACTERIZATION OF THE COLONIZATION FACTOR ANTIGEN II PLASMID (CFA/II) FROM ENTEROTOXIGENIC ESCHERICHIA COLI

The etiological role of enterotoxigenic E. coli (ETEC) in diarrheal diseases of man and domestic animals is firmly established. Besides the production of enterotoxins (ST and LT), ETEC produces other important virulence factors; the colonization factor antigens (CFAs). CFAs mediate the attachment of ETEC to the epithelial cells of the small intestine, and this favors colonization by the bacteria and facilitates delivery of the enterotoxins to the intestinal cells.^ The production of enterotoxin and CFA is determined by plasmids and has been found to be restricted to a select number of E. coli serotypes.^ In this work, plasmid DNA analysis was performed in twenty-three CFA/II-producing enterotoxigenic Escherichia coli strains and their spontaneous CFA/II-negative derivatives. In some cases, strains lost the high molecular weight plasmid and also the ability to produce CFA/II, ST and LT. In other cases there was a deletion of the plasmid, which produced strains that were CFA/II('-), ST('-), LT('-) or CFA/II('-), ST('+), LT('+).^ The CFA/II plasmid from strain PB-176 (06:H16:CFA/II('+), ST('+), LT('+)) was transferred by transformation into E. coli K12 with concomitant transfer of the three characteristics: CFA/II, ST and LT.^ A physical map of the prototype CFA/II:ST:LT (pMEP60) plasmid was constructed by restriction endonuclease analysis and compared to plasmids from three other CFA/II-producing strains. A CFA/II-negative (but ST and LT positive) deletion derivative of pMEP60 (pMEP30) was also included in the map. The four CFA/II plasmids analyzed had a common region of approximately 30 kilobase pairs. The toxin genes were approximately 5 kbp apart and about 20 kbp from the common region. The information given by this physical map could be of great value when constructing a clone that will express the CFA/II genes but not the toxin genes. ^

CHARACTERIZATION OF HUMAN ACID ALPHA-GLUCOSIDASE

The goal of this study was to investigate the properties of human acid (alpha)-glucosidase with respect to: (i) the molecular heterogeneity of the enzyme and (ii) the synthesis, post-translational modification, and transport of acid (alpha)-glucosidase in human fibroblasts.^ The initial phase of these investigations involved the purification of acid (alpha)-glucosidase from the human liver. Human hepatic acid (alpha)-glucosidase was characterized by isoelectric focusing and native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four distinct charge forms of hepatic acid (alpha)-glucosidase were separated by chromatofocusing and characterized individually. Charge heterogeneity was demonstrated to result from differences in the polypeptide components of each charge form.^ The second aspect of this research focused on the biosynthesis and the intracellular processing and transport of acid (alpha)-glucosidase in human fibroblasts. These experiments were accomplished by immune precipitation of the biosynthetic intermediates of acid (alpha)-glucosidase from radioactively labeled fibroblasts with polyclonal and monoclonal antibodies raised against human hepatic acid (alpha)-glucosidase. The immune precipitated biosynthetic forms of acid (alpha)-glucosidase were analyzed by SDS-PAGE and autoradiography. The pulse-chase experiments demonstrated the existence of several transient, high molecular weight precursors of acid (alpha)-glucosidase. These precursors were demonstrated to be intermediates of acid (alpha)-glucosidase at different stages of transport and processing in the Golgi apparatus. Other experiments were performed to examine the role of co-translational glycosylation of acid (alpha)-glucosidase in the transport and processing of precursors of this enzyme.^ A specific immunological assay for detecting acid (alpha)-glucosidase was developed using the monoclonal antibodies described above. This method was modified to increase the sensitivity of the assay by utilization of the biotin-avidin amplification system. This method was demonstrated to be more sensitive for detecting human acid (alpha)-glucosidase than the currently used biochemical assay for acid (alpha)-glucosidase activity. It was also demonstrated that the biotin-avidin immunoassay could discriminate between normal and acid (alpha)-glucosidase deficient fibroblasts, thus providing an alternative approach to detecting this inborn error in metabolism. (Abstract shortened with permission of author.) ^

REGULATION OF CYTOSKELETAL ASSEMBLY: A STUDY OF THE INTERACTION OF FILAMIN AND F-ACTIN

Filamin is a high molecular weight (2 x 250,000) actin crosslinking protein found in a wide variety of cells and tissues. The most striking feature of filamin is its ability to crosslink F-actin filaments and cause ATP-independent gelation and contraction of F-actin solutions. The gelation of actin filaments by filamin involves binding to actin and crosslinking of the filaments by filamin self-association. In order to understand the role of filamin-actin interactions in the regulation of cytoskeletal assembly, two approaches were used. First, the structural relationship between self-association and actin-binding was examined using proteolytic fragments of filamin. Treatment of filamin with papain generated two major fragments, 90Kd and 180Kd. Upon incubation of the papain digest with F-actin and centrifugation at 100,000 x g, only the 180Kd fragment co-sedimented with F-actin. The binding of the 180Kd fragment, P180, was similar to native filamin in its sensitivity to ionic strength. Analytical gel filtration studies indicated that, unlike native filamin, P180 was monomeric and did not self-associate. Thermolysin treatment of P180 produced a 170Kd fragment, PT170, which no longer bound and co-sedimented with F-actin. These results suggested that filamin contained a discrete actin-binding domain. In order to locate the actin-binding domain, affinity purified antibodies to the papain and thermolysin sensitive regions of filamin were used in conjunction with filamin fragments generated by digestion with S. aureus V8 protease and elastase. The results indicated that the papain and thermolysin cleavage sites were close together, and, most likely, within 10Kd of one another. Taken together, these data suggest that filamin contains a discrete, internal actin-binding domain. The second approach was to use the non-crosslinking fragment P180 to develop a quantitative assay of filamin-actin binding. The binding of ('14)C-carboxyalkylated P180 was examined using the co-sedimentation assay. ('14)C-P180 binding to actin was equivalent to that of unlabelled P180 and exhibited comparable sensitivity of binding to changes in ionic strength. Within 5 min. of incubation the process had reached equilibrium. The specificity of binding was shown by the lack of binding of ('14)C-PT170. The binding of ('14)C-P180 was found to be a reversible and saturable process, with a K(,d) of 2 x 10('-7) M. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^