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Biomedical subjects

P J Knight

Publications and source records attributed to P J Knight.

At least 19 recordsLinked to original sources
PubMed1996

Comparison of reflectance and transmission densitometry, using document and laser scanners, for quantitation of stained Western blots.

The quantitation of stained Western blots by reflectance and transmission scanning was explored using a blot of monoclonal immunoglobulin G probed with anti-mouse antibody. The so-called "scanned absorbance" output from a document scanner was found to be directly proportional to the fraction of light absorbed rather than obeying the logarithmic relationship expected for true spectrophotometric absorbance. This explains observations in the literature of a strongly curved relation between loading and "absorbance." Laser transmission densitometry of a blot immersed in a clarifying solvent mixture showed that peak area was linearly related to loading over a wide range. In reflectance mode the document scanner also gave equally linear quantitation of dry blots, providing that a logarithmic correction curve was applied during scanning. It was found advantageous to interpose a red acetate filter sheet between the blot and the scanning table to aid detection of weakly stained bands. The document scanner gave less satisfactory results when used in transmission mode on a clarified blot because weak bands were poorly quantitated.

Animals↗
PubMed1996

Clarification of immunoblots on polyvinylidene difluoride (PVDF) membranes for transmission densitometry.

The opacity of polyvinylidene difluoride (PVDF) transfer membrane can be reduced by infiltration with liquid. A mixture of 9:1 v/v ethylene glycol/glycerol gives complete transparency; clarification by this mixture is stable due to the low volatility of the components and is reversed by washing in water without loss of band intensity. The mixture is easy to apply to the membrane and being water soluble, non-corrosive and relatively non-toxic, is convenient to use. Clarification and densitometric analysis of stained immunoblots of PVDF membranes is at least as effective as that previously described for nitrocellulose, but the greater ease of use of the new mixture makes PVDF the membrane of choice for transmission densitometry. Using a blot onto PVDF of monoclonal IgG probed with anti-mouse antibody, laser transmission densitometry revealed that the stained peak area was related to loading in a linear fashion over a 60-fold range on a single blot.

Animals↗
PubMed1996

Dynamic behaviour of the head-tail junction of myosin.

It has generally been supposed that flexibility in the head-tail junction of myosin is provided by free rotation around the bonds of the polypeptide backbone of a few amino acid residues, but direct evidence for this is lacking. Here it is shown that the binding of an antibody in this region reveals a novel structure in which the bases of the heads are separated by 10 nm, with concomitant 9 nm shortening of the tail and movement of the sites of sharp bends in the tail a similar distance towards the heads. These results suggest that the junction is a dynamic structure in which between 60 and 130 residues of the coiled coil can separate to allow the heads to move apart. They suggest a site for the series elastic element of the cross-bridge, and have implications for the interactions of the two heads with actin subunits in the thin filaments, and the mechanism of movement of other two-headed motor proteins, such as kinesins.

Actins↗
PubMed1995

Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin.

The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue pre-pro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.

Amino Acid Sequence↗
PubMed1994

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N.

A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

Amino Acid Sequence↗
PubMed1993

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.

Animals↗
PubMed1991

N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis.

Proteins synthesized by the bacterium Bacillus thuringiensis are potent insecticides. When ingested by susceptible larvae they rapidly lyse epithelial cells lining the midgut. In vitro the toxins lyse certain insect cell lines and show saturable, high-affinity binding to brush-border membrane vesicles (BBMVs) prepared from insect midguts. We observed that the sugar N-acetyl galactosamine (GalNAc) specifically decreased the cytolytic activity of a CryIA(c) toxin towards Choristoneura fumiferana CF1 cells, completely abolished toxin binding to Manduca sexia BBMVs, partially inhibited binding to Heliothis virescens BBMVs and had no apparent effect on binding to Pieris brassicae BBMVs. In ligand blotting experiments the toxin bound proteins of 120 kDa in M. sexta, 125 kDa in P. brassicae and numerous proteins in H. zea. Toxin binding to these proteins was specifically inhibited by GalNAc. The toxin binding proteins of M. sexta and H. zea also bound the lectin soybean agglutinin. Taken together these findings suggest that N-acetyl galactosamine might be a component of a CryIA(c) toxin receptor of CF1 cells and of at least two of the insects tested.

Acetylgalactosamine↗
PubMed1988

Transmission densitometry of stained nitrocellulose paper.

We report a simple solution to the problem of quantitative densitometry of stained nitrocellulose paper. By immersing the paper in a household lubricating oil of matching refractive index, the light-scattering properties of the paper are largely eliminated, allowing precise transmission densitometry in any flat bed densitometer. The method was evaluated on immunochemically stained Western blots of the proteinases cathepsins B and L. An approximately linear relationship was found between the integrated absorbance of the stained zone and the logarithm of the amount of protease applied to the polyacrylamide gel over the range of 150 to 350 ng of cathepsin B and 50 to 250 ng of cathepsin L.

Blotting, Western↗
PubMed1988

Studies of IgG, IgM and IgA antiphospholipid antibody isotypes in systemic lupus erythematosus.

We evaluated the clinical relevance of 6 antiphospholipid antibodies including cardiolipin and their IgG, IgM and IgA isotypes in 92 patients with systemic lupus erythematosus (SLE). Antiphospholipid antibodies generally had significant associations with thrombocytopenia and a history of false-positive syphilis serologies. In 4 of 6 antiphospholipid antibodies, an inverse association with renal disease was observed. Antiphospholipid antibodies may moderate or protect against renal disease, or this may reflect the high doses of corticosteroids and cytotoxic drugs received by this group. Further studies are needed to determine how many antibody families cause these activities and to elucidate whether certain SLE subgroups possess differing specificities for each of the phospholipids.

Adolescent↗
PubMed1987

Splenic lymphangiomatosis: a rare cause of splenomegaly.

A case of lymphangiomatosis of the spleen and adrenal in a 10-year-old girl is described. The patient had multiple recurrent lymphangiomata excised from her neck during the first 4 years of life. She presented with a recurrent cystic hygroma of the neck and was found to have an enlarged spleen. This increased in size during 5 months of observation, causing left upper quadrant discomfort, and was excised. The diagnosis, management, and prognosis of this rare disorder are discussed.

Adrenal Gland Neoplasms↗
PubMed1986

Distribution of mass within native thick filaments of vertebrate skeletal muscle.

The distribution of mass within the vertebrate skeletal thick filament has been determined by scanning transmission electron microscopy. Thick and thin filaments from fresh rabbit muscle were mixed with tobacco mosaic virus (TMV), fixed with formaldehyde, dried onto thin carbon films and viewed in a computer-linked microscope. Electron scattering data from both TMV and thick filaments were analysed with reference to the long axis of the particles so that the distribution of mass within the particles could be determined. While TMV appeared to be a uniform rod at the resolution employed (4.3 nm), the thick filament was clearly differentiated along its length. M-line remnants at the centre of the filament were flanked by regions of low mass per unit length, corresponding to the bare zone of the filament, and then by the more massive cross-bridge regions. The mass per unit length was approximately constant through most of the cross-bridge zone and declined at the filament tips, in a manner consistent with a constant number of myosin molecules per 14.3 nm interval (crown) throughout the cross-bridge zone. Fourier analysis of the data failed to detect the expected 43 nm periodicity of C-protein. The total mass of the thick filament was 184 Mdalton (s.e.m., 1.6 X 10(6); n = 70). The mass of adhering M-line proteins was highly variable but, on average, was about 4 Mdalton. The total mass of the filament and the mass distribution in the cross-bridge zone are consistent with three myosin molecules per crown.

Actin Cytoskeleton↗
PubMed1986

The use of interstitial radiation therapy in the treatment of persistent, localized, and unresectable cancer in children.

Two children with cancer that persisted after multiple exploratory laparotomies, external beam radiation therapy, and multidrug chemotherapy had gold 198 (198Au) seeds implanted into their localized but unresectable tumor. Both children are alive, are receiving no therapy, and are disease-free more than 2 years later. These two cases indicate the value of interstitial implant therapy in the treatment of some children with cancer.

Abdominal Neoplasms↗
PubMed1984

The diagnosis and treatment of the acute scrotum in children and adolescents.

Diagnosing acute scrotal pain and swelling in children and adolescents is urgent and often difficult. A review of 395 boys hospitalized with acute scrotal pain and/or swelling shows that a useful approach is to divide these patients into four groups--those with intermittent but recurrent episodes of pain, those with pathognomonic physical findings, those with definite epididymitis, and a remaining group with nonspecific swelling and tenderness. Five per cent of boys in this series presented with recurring episodes of scrotal pain; these boys should undergo a simple scrotal operation that yields excellent results. Eight per cent had pathognomonic physical findings; treatment in these boys is straightforward. Eighteen per cent had a definite diagnosis of acute epididymitis (i.e., three nonpathognomonic but suggestive findings of acute epididymitis or two suggestive findings plus a radionuclide scan showing bilateral perfusion); nonoperative therapy is indicated in this group. In the remaining boys, scrotal exploration is the diagnostic (and usually therapeutic) procedure of choice.

Acute Disease↗
PubMed1983

The diagnosis and treatment of midline neck masses in children.

Frustration in differentiating before operation among the various causes of midline neck masses stimulated us to review the clinical, operative, and pathologic findings in 176 children hospitalized for a midline cervical lump. The preoperative diagnosis was correct in 61% of these cases, the surgeon's postoperative diagnosis was correct in 83%, and the pathologist's diagnosis was correct in 98%. Only recurrence following prior excision and persistent sinus following drainage of a midline cyst were pathognomonic preoperative indicators of a thyroglossal duct cyst; these findings were present in only 6% of the cases. The age of the child, a history of inflammation or of fluctuation in size, the dimensions of the mass, and the relationship of the mass to the hyoid were useful in differentiating among groups of children with a thyroglossal cyst, an epidermoid cyst, or an enlarged perihyoid lymph node. The duration of the presence of the mass and its movement with swallowing were not helpful. Using the preoperatively recorded historical and physical findings in these children with a known cause for their midline neck mass and applying Bayes' theorem, one can only achieve a correct preoperative diagnosis with an 80% accuracy based on the best available data. Consequently, additional information obtained at surgical exploration is needed to establish a secure diagnosis and to ensure appropriate treatment.

Adolescent↗
PubMed1983

Superficial lumps in children: what, when, and why?

Deciding whether a visible or palpable lump should be excised in a trivial problem if one believes that every unexplained mass in an infant or a child must be promptly removed. But with the present increased public awareness of cancer, this approach would unnecessarily raise parental anxiety. This review of superficial lumps excised in infants and children shows that approximately 1% are malignant. Although a precise diagnosis on the basis of clinical findings remains imperfect, four fifths of the malignant lesions can be recognized on the basis of five risk factors: onset in the neonatal period, a history of rapid or progressive growth, skin ulceration, fixation to or location deep to the fascia, and a firm mass greater than 3 cm in diameter. In the absence of any of these risk factors, parents can be reassured with a 99.7% accuracy about the benign nature of their child's lump at the initial consultation. Approximately 6% of these lumps will spontaneously regress and, therefore, do not require excision. However, more than 90% of superficial lumps will persist or slowly enlarge and should be electively excised for cosmetic reasons, to prevent late infection or inflammation, and to diagnose the remaining three malignant lesions per thousand lumps that would not be recognized using the above five risk factors.

Adolescent↗