Biomedical subjects
Publications and source records attributed to M Huber.
A probe holder for long-term measurements of intranasal microcirculation by laser Doppler flowmetry is described. It is adjustable to any physiognomy and allows precise intranasal probe insertion. It is based on a commercially-available shooting-spectacles frame and might also be useful for other measurements such as intranasal temperature, humidity and pO2.
Health care delivery institutions and providers, employers, and government agencies throughout the U.S. are sharing information and pooling resources in an attempt to produce reliable measurements of health outcomes. The rapid rate of growth in the collection and pooling of health outcomes data has prompted the need for standardization. The work of health care organizations and consortiums pooling data would be greatly facilitated by widely accepted standards for the coding and transmitting of outcomes data. Moreover, standards allow for the inter-operation of data capture products, data analysis tools, and data pooling services developed by a variety of different vendors. The Health Outcomes Institute (HOI) and Henry Ford Health System (HFHS) have developed and maintain a database of health outcomes questions which provides a mechanism for uniquely coding data elements. HFHS and the American Medical Group Association (AMGA) have created a software tool to facilitate the conversion and transmission of health outcomes data elements in an American Society for Testing and Materials (ASTM)/Health Level Seven (HL7) format, which incorporates HOI question standards.
A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.
Spermine is often the most abundant polyamine in human tumors such as breast carcinomas. However, its specific role in tumor biology is still uncertain, since inhibitors of ornithine decarboxylase such as alpha-difluoromethylornithine depress cell growth while leaving spermine content mostly unaffected. We have assessed the specific role of spermine in breast cancer cell growth using N-cyclohexyl-1,3-diaminopropane (C-DAP), a potent spermine synthase inhibitor. In ZR-75-1 cells, C-DAP decreased net cell growth after 14 days by 65% at 50 microm, with an IC50 of about 5 microM, and was about 10 times more potent than N-(n-butyl)-1,3-diaminopropane, another spermine synthase inhibitor. C-DAP acted as a specific inhibitor of spermine biosynthesis, since (a) it depleted spermine content while causing an equal or greater accumulation of spermidine on a molar basis, (b) it rapidly induced S-adenosylmethionine decarboxylase activity and the accumulation of its products due to relief of spermine-dependent inhibition of enzyme expression, and (c) exogenous spermine (1 microM) completely reversed C-DAP-induced growth inhibition. C-DAP and related compounds were accumulated, at least in part, through a mechanism distinct from the polyamine transport system, while also blocking putrescine and spermidine uptake with various potencies. Reversibility of C-DAP-induced growth inhibition by exogenous spermine was progressively lost on prolonged treatment, in association with marked morphological changes. In 4 different human breast cancer cell lines (ZR-75-1, T47-D, MCF-7, and MDA-MB-231), relative growth sensitivity to C-DAP was inversely related to the extent of spermidine accumulation caused by spermine synthase inhibition, suggesting that spermidine overaccumulation can functionally replace spermine. Interestingly, C-DAP strongly potentiated growth inhibition caused by alpha-difluoromethylornithine in all cell lines tested by preventing conversion of residual spermidine to spermine, indicating that spermine synthesis limits alpha-difluoromethylornithine action and that under some critical threshold, spermidine cannot fulfill cellular needs for spermine. Thus, spermine plays specific and important functions in breast tumor growth, and spermine synthase inhibitors could markedly improve the therapeutic effectiveness of existing polyamine depletion strategies, especially in spermine-rich tumors.
Lamellar ichthyosis is a severe congenital skin disorder characterized by generalized large scales and variable redness. Affected individuals in three families exhibited drastically reduced keratinocyte transglutaminase (TGK) activity. In two of these families, expression of TGK transcripts was diminished or abnormal and no TGK protein was detected. Homozygous or compound heterozygous mutations of the TGK gene were identified in all families. These data suggest that defects in TGK cause lamellar ichthyosis and that intact cross-linkage of cornified cell envelopes is required for epidermal tissue homeostasis.
Dystonia is characterized by slow, repetitive, involuntary, often twisting movements leading to sustained abnormal postures and can be focal or generalised. In most cases, etiology remains unrevealed, some cases are hereditary, others have metabolic origin or can be attributed to focal brain lesions. The most common types of dystonia, spasmodic torticollis and blepharospasm, can now be treated successfully, albeit symptomatically, with Botulinum toxin injections into the affected muscles. A practical guide to differential diagnostic and therapeutic strategies is presented.
The murine CD44 receptor family is thought to be involved in a variety of lymphocyte functions, including lymphopoesis, lymphocyte homing and cell migration. Herein, we show that murine CD44 also plays a role as a co-stimulatory molecule for the activation of CD4+ T cells. Ligation of CD44 by mAb enhanced IL-2 production of long-term cultured, anti-CD3-stimulated Th1 cell lines. Moreover, anti-CD44 mAb synergized with anti-CD28 mAb in exerting this effect. A synergism of anti-CD28 and anti-CD44 mAb to co-stimulate IL-2 production was also observed in anti-CD3-triggered, freshly isolated splenic CD4+ T cells. Blocking experiments with cyclosporin A indicated that the intracellular pathways used by the CD28 and CD44 molecules appear to be different. In contrast to the effects on the IL-2 production of Th1 cells, neither anti-CD44 mAb alone nor the combination of anti-CD44 with anti-CD28 were able to induce proliferation of anti-CD3-triggered Th1 cells. In accordance, triggering of CD44 and/or CD28 by mAb was not sufficient to reverse the previously described 'proliferative block'. This term describes the unresponsiveness of Th1 cells against IL-2, which occurs when Th1 cells are triggered by anti-CD3 in the absence of co-signals. These data lead us to propose a model of Th1 cell activation which includes two functionally different types of co-signals: one for IL-2 production and a separate one for proliferation.
We recently identified mutations of the keratinocyte transglutaminase gene as a cause of lamellar ichthyosis. In this study we analyzed two sporadic cases of lamellar ichthyosis. Transglutaminase activity measured in membrane extracts from cultured differentiating keratinocytes was within the range observed in normal individuals. Western blot and Northern blot analysis revealed normal size and quantities of keratinocyte transglutaminase protein and mRNA. Sequencing of the 15 exons and their flanking regions demonstrated no deviation from the published sequence except for two silent polymorphisms. These results exclude mutations of keratinocyte transglutaminase as a cause for lamellar ichthyosis in these patients, indicating that lamellar ichthyosis is a genetically heterogeneous disorder.
Loricrin, involucrin, small proline-rich protein (SPRR)1, SPRR2, and SPRR3 genes are located within a cluster of 1.5 Mbp on chromosome 1q21 and most likely evolved from a common ancestor. Monospecific polyclonal antibodies and cDNA probes were produced to investigate SPRR transcripts and proteins. SPRR expression was restricted to terminally differentiating squamous cells, preferentially located at the cell periphery, and immunoreactivity was greatly reduced in cells with a mature cornified cell envelope. Furthermore, detectable SPRR2 and SPRR3 levels were strongly increased in differentiating keratinocyte cultures after addition of LTB-2, a specific inhibitor of transglutaminases, suggesting that they are precursor proteins of the cornified cell envelope. In normal epidermis, SPRR1 was restricted to appendageal areas, SPRR2 was expressed coherently, and SPRR3 was completely absent. In the upper digestive tract, SPRR1 was expressed in sublingual and tongue epithelium, SPRR2 was mostly restricted to lingual papillae, and SPRR3 was abundant in oral and esophageal epithelium. In psoriatic epidermis, SPRR1 and SPRR2 were expressed at much higher levels than in normal epidermis. Addition of 10(-7) M retinoic acid to cultured differentiating keratinocytes significantly down-regulated the expression of SPRR2 and SPRR3 transcripts and slightly decreased that of SPRR1. Thus, SPRR1, SPRR2, and SPRR3 are differentially expressed in vivo and in vitro, suggesting that the SPRR multigene family evolved to serve as highly specialized cornified cell envelope precursor proteins in stratified epithelia.
Epidermolytic palmoplantar keratoderma is an autosomal dominant skin disorder characterized by hyperkeratosis of the palms and soles. Ultrastructurally the disease exhibits abnormal keratin filament networks and tonofilament clumping like that found in the keratin disorders of epidermolysis bullosa simplex and epidermolytic hyperkeratosis. The disease has been mapped to chromosome 17q11-q23 in the region of the type 1 keratin gene locus and more recently mutations have been found in the palmoplantar specific keratin, keratin 9. We have analyzed six unrelated incidences of epidermolytic palmoplantar keratoderma for mutations in their keratin 9 genes. In two of these, we have identified mutations that alter critical residues within the highly conserved helix initiation motif at the beginning of the rod domain of keratin 9. In a three-generation Middle Eastern kindred we found a C to T transition at codon 162 that results in an arginine to tryptophan substitution at position 10 of the 1A alpha-helical domain, thus confirming this codon as a hot spot for mutation in keratin 9. The other mutation found involves a T to C transition at codon 167 that results in the expression of a serine residue in place of the normal leucine at position 15 of the 1A segment and is the first documentation of this mutation in this gene. The identification of these substitutions extends the current catalog of disease causing mutations in keratin 9.
Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB.
In a retrospective study of women who had undergone pelviscopic hysterectomies in the last 2 years, we focused on postoperative cyclic spotting. Preoperatively, a regular, cyclic menstruation and a premenopausal sexual hormone constellation were common to all patients. In the first group (n = 29), spotting occurred only in association with two uterine anatomical varieties: uterine hyperflexion and cervical elongation. The second group (n = 18), in which intraoperative internal electrocoagulation of the residual cervical envelope was performed, no spotting occurred despite the presence of uterine hyperflexion and cervical elongation. This procedure prevents the occurrence of postoperative spotting in cases of uterine anatomical varieties.
PURPOSE: We conducted a phase II study to determine the response to and toxicity of docetaxel (Taxotere; Rhône-Poulenc Rorer Pharmaceuticals, Inc, Collegeville, PA) in patients with advanced non-small-cell lung cancer refractory to prior platinum-containing chemotherapy (PCC) regimens. PATIENTS AND METHODS: Forty-four patients with stage IIIb or IV platinum-refractory non-small-cell lung cancer were treated with 100 mg/m2 of docetaxel intravenously over 1 hour every 3 weeks. The responses of 42 of 44 patients were assessable. Most patients had a Zubrod performance status of 1; the predominant histologic type was adenocarcinoma (61%), and 91% of patients had stage IV disease. RESULTS: Nine of 42 assessable patients (21%) achieved a partial response to treatment. The median response duration (from response to progression) was 17 weeks, and the projected median survival duration of all patients is 42 weeks (51 weeks for adenocarcinoma and 22 weeks for nonadenocarcinoma). Grade 3/4 neutropenia occurred in 85% of patients and was associated with fever that required intravenous antibiotics in 16% of patients (3% of cycles). Other acute side effects included easily treated hypersensitivity reactions and dermatitis. Cumulative side effects included fluid retention and neuropathy. CONCLUSION: Docetaxel administered at 100 mg/m2 intravenously every 3 weeks has notable activity against platinum-refractory non-small-cell lung cancer, with a 21% major response rate. Primary side effects were neutropenia, hypersensitivity, and fluid retention.
The separation of time and space in processing a sample greatly simplifies the design of automation for clinical testing. The efficient spatial arrangement of analytical units and sample manipulators has become a more complex task because of the degree of automation required on today's state-of-the-art analyzer. Minimization of sample volume and the reduction of overall analyzer size further complicate the design problem. We report the development of a proprietary method of decoupling the temporal and spatial elements required for analysis of samples. This process is based on number theory and can be used to optimize the distance between the physical processing stations while allowing these same stations to operate on samples over a substantial range of times. The technique is versatile and can also be used when it is desirable to sequentially move groups of items from location to location.
Subacute sclerosing panencephalitis (SSPE), a very rare but lethal disease caused by measles viruses (MV) persisting in the human central nervous system (CNS) is characterized by lack of viral budding, reduced expression of the viral envelope proteins and spread of MV genomes through the CNS despite massive immune responses. The five major MV genes from several SSPE cases were cloned and sequenced, the two transmembrane envelope glycoproteins hemagglutinin (H) and fusion protein (F) were expressed and their maturation, cellular localization and functionality analyzed. We conclude that 1) mutations in the MV genes arise not only individually, by errors of the MV polymerase, but also in clusters as hypermutations, presumably due to RNA unwinding/modifying activity altering accidentally formed double-stranded RNA regions, 2) MVs spread in SSPE brains after clonal selection, 3) the MV matrix (M) gene is most heavily mutated and dispensable, 4) the two genes encoding envelope transmembrane proteins give rise to functional but altered proteins (typically F is heavily altered in its cytoplasmic domain), 5) H protein is transported poorly to the cell surface, 6) F and H proteins maintain tightly interdepending fusion functions, presumably to allow local cell fusion and MV ribonucleoprotein (RNP) spread through the CNS.
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Seven site-directed mutants of the bacterial photosynthetic reaction center (RC) from the 2.4.1 and WS 231 wild-type strains of Rhodobacter sphaeroides have been crystallized and their X-ray diffraction analyzed to resolutions between 3.0 and 4.0 A. The mutations can be divided into four distinct categories: (1) mutations altering cofactor composition that affect electron transfer and quantum yield, His M202-->Leu (M202HL), His L173-->Leu (L173HL), and Leu M214-->His (M214LH); (2) a mutation in the proposed pathway of electron transfer altering electron-transfer kinetics, Tyr M210-->Phe (M210YF); (3) a mutation around the non-heme iron resulting in an iron-less reaction center, His M219-->Cys (M219HC); and (4) mutations around the secondary electron acceptor, a ubiquinone, affecting proton transfer and quinone turnover, Glu L212-->Gln (L212EQ) and Asp L213-->Asn (L213DN). Residues L173 and M202 are within bonding distance of the respective magnesiums of the two bacteriochlorophylls of the BChl special pair, while M214 is close to the bacteriopheophytin on the active A branch of the RC. The L173HL and M202HL crystal structures show that the respective bacteriochlorophylls are replaced with bacteriopheophytins (i.e., loss of magnesium) without significant structural perturbations to the surrounding main-chain or side-chain atoms. In the M214LH mutant, the bacteriopheophytin has been replaced by a bacteriochlorophyll, and the side chain of His M214 is within ligand distance of the magnesium. The M210YF, L212EQ, and L213DN mutants show no significant tertiary structure changes near the mutation sites. The M219HC diffraction data indicate that the overall tertiary structure of the reaction center is maintained in the absence of the non-heme iron.