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Discriminant functions.

Discriminant Functions (DFs), first described by Fisher in 1936, have been applied to the classification of microcytic disorders such as iron deficiency and heterozygous thalassemia. Mathematically DFs are weighted linear combinations of variables. If the underlying assumption of multivariate normality is valid DFs provide the best possible classification. Variables may need to be transformed before the DF is derived. When two groups have to be classified it is easy to visualise the DF. With one variable the DF is represented by the point which provides the best separation. In the bivariate situation the two groups form ellipses and the DF is the best line of separation whilst in the trivariate case the two groups are ellipsoids and a plane forms the best separation. Ratios and power functions are equivalent to DFs but they are less efficient and less rigorously derived. To apply DFs in hematological practice it is necessary to carefully select the measurements to be included and to define the case selection criteria. Once the DF has been derived it should be tested on a new data set and its transferability assessed. Like any single test the DF will have sensitivity and specificity which may need to be adjusted by changing the "cut-off" if the DF is used for screening rather than for differential diagnosis.

Diagnosis

Linkage studies of X-linked mental retardation: high frequency of recombination in the telomeric region of the human X chromosome (fragile site/linkage/recombination/X chromosome).

One of the commonest forms of X-linked mental retardation is associated with a fragile site at Xq27 on the human X chromosome which can be visualised structurally after culturing cells in folate-deficient media. Unusually, the mutation can be transmitted through a phenotypically normal male. There is already some evidence that the gene loci for G6PD and factor IX are linked to this mental retardation locus. We have followed the inheritance of a DNA sequence 52A, in fragile site families that are also informative for factor IX. We demonstrate that these probes are localised at Xq27/Xq28-Xqter, close physically to the fragile site. We did not find close linkage between 52A, factor IX, and the fragile site in the families studied despite 52A and factor IX showing linkage in normal families. We discuss the importance of these data for the genetic mapping of this region of the human X chromosome and the implication for the use of these DNA probes for clinical diagnosis.

Animals

Visualisation by low-angle shadowing of the leucocyte-common antigen. A major cell surface glycoprotein of lymphocytes.

The leucocyte-common antigen (L-CA) from rat thymocytes is a cell surface glycoprotein of 180 000 apparent mol. wt. with an 80-kd cytoplasmic domain. This paper reports the molecular dimensions of the molecule visualised by electron microscopy after low-angle shadowing. The L-CA monomer consists of a globular head region of approximately 12 nm diameter and a short tail approximately 18 nm long. In deoxycholate both monomers and multimers are seen with aggregation occurring at the head groups. When the detergent is removed, larger clusters are formed with tails extending from a central aggregate. A 100-kd tryptic fragment of L-CA that is known to include the extracellular parts of the molecule also exists in monomer and multimer forms and is seen to have a rod-like structure of length 28 nm without evidence of the head group. Altogether the data indicate that the rod-like structure is found outside the cell and that the extra sequence that forms the head is inside. The tryptic fragment is likely to be derived by cleavage after the transmembrane sequence.

Animals

Alterations of CAP audiogram by increased endolymphatic pressure and its relation to hydrops.

Most current theories regarding the inner ear pathology of Menières disease assume that there is an augmentation of the endolymphatic pressure due to the presence of hydrops. In this study normal hearing pigmented guinea pigs were employed to investigate the effect of increased endolymphatic pressure on the compound action potential (CAP) audiogram. All animals were implanted with an electrode on the round window and the CAP audiogram was determined prior to further surgery. The endolymphatic canal was then visualised by a posterior fossa intra-dural surgical approach. A hole was pierced in the canal and a cannula inserted. The CAP audiogram was again determined before, and at frequent intervals after, the application of hydrostatic pressure (0.5-1 cm Hg). A similar sequence of CAP sensitivity losses was observed within 2 h for 0.5 cm Hg or 15 min for 1 cm Hg. There was at first a very high frequency loss, followed by a very low frequency loss and finally a mid frequency sensitivity loss rendered the audiogram flat and lying around 50 dB sound pressure level. Given that the first characteristic index for experimental hydrops is a low frequency loss the present data suggest that an increase in endolymphatic pressure, as in these experiments, is likely to be a rather late pathological feature of hydrops. Indeed we have shown that a high frequency loss develops at a second phase during the evolution of hydrops.

Action Potentials

A novel protein antigen of the malaria parasite Plasmodium falciparum, located on the surface of gametes and sporozoites.

A Plasmodium falciparum cDNA clone was isolated of which the insert is transcribed at high rates as a 1.4-kb mRNA in the sexual stages of the malaria parasite. The cDNA clone contains a copy of a non-interrupted gene which codes for a protein of 157 amino acids (Mr = 16607). This 16-kDa protein does not contain repetitive sequences and is characterised by a putative N-terminal signal sequence, a hydrophobic membrane anchor sequence and a highly hydrophilic C-terminal region suggesting that it is an integral membrane protein. Rabbit antisera raised against a synthetic peptide covering amino acids 31-47 of the 16-kDa protein and against recombinant fusion proteins recognised the 16-kDa antigen in protein extracts of gametocytes, macrogamete/zygotes and sporozoites by Western blot analysis. The rabbit antisera also reacted with gametes, gametocytes and sporozoites in a standard immunofluorescence assay. By immunoelectron microscopy using the protein A-gold method the 16-kDa protein could be clearly visualised on the surface of macrogametes and sporozoites, whereas the antigen was not detectable in the asexual erythrocytic stages of the parasite. The 16-kDa antigen of P. falciparum therefore might have the potential to elicit a dual protective immune response against the sporozoite and sexual stage parasites.

Amino Acid Sequence

Laparoscopic laser cholecystectomy: our first 200 patients.

A series of 200 consecutive patients were considered for laparoscopic laser cholecystectomy. Laparoscopic laser cholecystectomy was attempted in 195 cases and was performed in 192 cases. Laparoscopy was performed in five patients, but laparoscopic cholecystectomy was not attempted owing to dense adhesions (3), cholangiocarcinoma (1) and an absent gallbladder (1). The indications for operation were symptomatic gallstones which included biliary colic (142), acute cholecystitis (49) and gallstone pancreatitis (9). The median duration of operation was 75 min. Operative cholangiography was attempted in 151 (77%) of cases, and was successful in 85% of attempts. Laparoscopic common bile duct visualisation was performed three times with successful stone extraction twice. The other common bile duct was normal. The median duration of postoperative hospital stay was 2 days, for return to normal activity 6 days, and for return to work 10 days. Mean analgesic and antiemetic requirements were approximately one-third of those for open cholecystectomy. Of the patients, 94% reported good or excellent overall satisfaction and 96% reported excellent cosmetic results. Seven complications occurred (4%). Three patients had immediate conversion to laparotomy owing to haemorrhage (2) and gallbladder rupture (1). Four patients required laparotomy for postoperative complications (common bile duct damage, slipped clips from cystic duct, perforated duodenum and leaking accessory hepatic duct). No complications occurred in the last 140 cases. These data suggest that laparoscopic laser cholecystectomy reduces the discomfort of laparotomy and allows a shorter postoperative recovery. The operation has a learning curve, but will ultimately be applicable to the majority of patients with symptomatic gallstones.

Adult

Immunohistochemical localisation of nerve growth factor in a subpopulation of chick spinal ganglion neurons.

We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Anti-sera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well-described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct.

Amino Acid Sequence

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase

Localization and function of the D3 dopamine receptor.

A novel dopamine receptor has been recently cloned, which differs from D1 and D2 receptors by its sequence, localization, pharmacology and possibly signalling system, hence its designation as D3 receptor. The D3 receptor cDNA was obtained by a combination of screenings of DNA libraries and PCR polymerase chain reaction experiments. It encodes a protein with a predicted structure consisting in 7 transmembrane domains indicating that it belongs to the G-protein coupled receptor family. Its global homology is 52% with the D2 receptor but 78% if only transmembrane domains are considered. Two shorter transcript variants, in addition to the full-length cDNA were detected by PCR in various rat brain regions. The shorter transcripts are generated by alternative splicing and encode two putative proteins respectively interrupted after the second transmembrane domain and lacking the second extracytoplasmic loop. After transfection of this latter isoform into cultured cells, no dopaminergic activity could be detected. These shorter splice variants may regulate the number of active D3 receptors. The human D3 receptor was also cloned using similar approaches with rat D3 receptor cDNA probes and was found highly homologous to the rat receptor, except in the third intracytoplasmic loop. The human D3 receptor gene was assigned to the chromosome 3 at q13.3 band. Visualisation of D3 receptor mRNA in rat brain by in situ hybridization indicated a predominant expression of the message in the ventral striatum and other "limbic" areas. There is no overlap in the distributions of D2 and D3 receptor mRNAs in discrete structures, suggesting that the two transcripts are expressed by different cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence

[2-dimensional echography in the preoperative evaluation of mitral stenosis].

2D echocardiography has become one of the most important investigations in the preoperative assessment of mitral stenosis. This study was undertaken to determine the reliability of the information so obtained, by comparison with the surgical appearances. The study population consisted of 104 patients (average age 45 years, 76% women) undergoing open heart surgery for pure mitral stenosis (72%) or mixed mitral valve disease (28%) between 1980 and 1981. All underwent 2D echo using a phased array Aloka SSD 800 80 degrees sector scanner. Cardiac catheterisation was performed in 102 cases and left ventricular angiography in 89 cases. The echocardiogramme was interpreted by an observer who had no knowledge of the surgical results. The mitral surface area, the condition of the valves and subvalvular apparatus and the predictive value of the possible surgical technique were analysed. The 2D echo mitral surface area was estimated by planimetry and quantitatively by using the Gorlin formula during catheterisation and by the surgical description preoperatively. 2D echo was more sensitive than M mode in the detection of severe mitral stenosis (90% vs 73%, p less than 0,01). The 2D echo-Gorlin correlation was quite good (R = 0,70, p less than 0,01) but was worse when the valves were very thickened. When compared with the surgical observations, 87% of the 2D echo data was correct. The thickness of the valves, their amplitude, the diastolic bowing of the anterior leaflet and the presence of calcification were assessed by 2D echo. The echo-surgical results matched perfectly in 76% of cases. The usual cause of error was underestimation of the degree of valvular damage. The valvular bowing and thickness were the most useful signs. Dense, brilliant echos of valvular calcification were found in only 58% of surgically proven cases of valvular calcification, but the error was often related to fine calcification, not visible on fluoroscopy, or to its localisation on the posterior leaflet. The subvalvular apparatus was evaluated in over 93% of patients, a complete study being possible in 73% of them. The 2D echo-surgical correlations were excellent in 90% of the cases in which it had been completely visualised. The chordal thickening was correctly predicted in 79% of cases. The surgical assessment was more pessimistic in 1/3 of cases in which the chordae appeared to be of normal thickness. The length of the chordae was correctly predicted in 68% of cases. The surgical assessment was more pessimistic in 1/2 of cases in which the chordae appeared to be of normal length.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult

Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA.

Rapid detection of deletion and duplication mutations that cause Duchenne and Becker muscular dystrophy was achieved in patients and carriers after amplification of small amounts of mRNA from peripheral blood lymphocytes. The entire coding region of the dystrophin mRNA was amplified in 10 sections by reverse transcription and nested polymerase chain reaction, and the products were directly visualised on acrylamide minigels with ethidium staining. Major structural gene mutations were identified by the appearance of a band of different size to that of the wild type. The altered band was readily detected in all patients and heterozygous relatives. This non-radioactive test of venous blood samples can be used for unambiguous and rapid identification of virtually all carriers of deletions or insertions within the dystrophin gene.

Base Sequence

Multiple mitochondrial DNA deletions in an elderly human individual.

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69-year-old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.

Aged

Natural course of Crohn's disease after ileocolic resection: endoscopically visualised ileal ulcers preceding symptoms.

Forty two Crohn's disease patients were followed up after ileocolic resection with regard to symptoms and endoscopic appearance of the ileocolic anastomosis. Twenty eight patients resected because of colonic neoplasm served as controls. In all the Crohn's disease patients the ileal resection margin was disease free macroscopically at operation. In addition, intraoperative ileoscopy was performed in 13 and no sign of residual inflammation in the neoterminal ileum was seen. Endoscopy soon after surgery often showed preanastomotic ileal ulceration before symptoms appeared, whereas no anastomotic lesions were observed in the controls. Thus, 22 of 30 Crohn's disease patients examined had ulceration of the anastomotic area after three months, but only 10 had developed symptoms indicating relapse (73 v 33%). Corresponding figures in the 30 patients examined after one year were 93 v 37%, and in 14 patients after three years they were 100 and 86% respectively. The inflammatory lesions in all cases were preanastomotic, in the neoterminal ileum, and showed time related progression from aphthae to larger ulcers and stricture. The study suggests that endoscopically observed inflammatory lesions that appear soon after ileocolic resection for Crohn's disease signify new inflammation and not residual, persistent disease or incomplete anastomotic healing. The data further suggest that despite clinical remission after apparently radical intestinal resection, the bowel is permanently inflamed in Crohn's disease.

Adolescent

Cross sectional echocardiographic assessment of great artery diameters in infants and children.

The pulmonary trunk and aortic root were measured on cross sectional echocardiograms in 173 normal subjects aged from one day to 15 years. Fifteen neonates were reexamined 3-6 days later. The great vessels were visualised in the parasternal long axis and short axis views. All measurements were made in end diastole and end systole by the leading edge method. The internal diameter (inner surface to inner surface) of the pulmonary trunk was also measured. The diameters of the great vessels correlated best with the square root of body surface area. Individual variability in cardiac growth gave a wide scatter of normal values. This was controlled for by calculating the ratio of the pulmonary trunk to aortic root for each subject. This ratio showed little individual variability and, except for the neonatal period, was remarkably constant throughout infancy and childhood (1.06 (0.06)). In the first 24 hours of life the ratio of the pulmonary trunk to the aortic root was significantly larger (1.29 (0.12)) but within one week it decreased to the "normal" ratio found in the older age groups. These normal data should be useful in assessing patients with congenital heart disease, particularly those in whom pulmonary blood flow is abnormal.

Adolescent

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension.

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.

Autoradiography

The major protein of bull seminal plasma: biosynthesis and biological function.

We isolated the major protein of apparent Mr of 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid respectively. Cell-free translation of poly(A)RNA from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent Mr of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Esch et al., Biochem. Biophys. Res. Comm. 113:861-867, 1983). The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP. Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a physiological process presumably related to the onset of sperm motility.

Amino Acid Sequence

Regulatory peptides of the gastrointestinal and respiratory tracts.

The gastrointestinal and respiratory tracts contain numerous regulatory peptides produced by and released from specialised epithelial cells and the organ innervation. This complex system of endocrine cells and nerves is generally called "the diffuse neuroendocrine system". Markers are now available which permit the visualisation of the diffuse neuroendocrine system or its individual components. These include antibodies to neuron-specific enolase, chromogranin, neurofilament triplet proteins, the brain protein S100 and antibodies to a variety of regulatory peptides. Peptides present in the gut and lung innervation include: vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), galanin, substance P, calcitonin gene-related peptide (CGRP), neuropeptide tyrosine (NPY), somatostatin and cholecystokinin (the latter two are also localised to endocrine cells of the gut). Bombesin-immunoreactivity is found in nerves in the gut and in endocrine cells of the foetal/neonatal lung. Neuropeptides of the gut and lung originate either from local neurons (e.g. VIP, PHI, galanin) or extrinsic neurons localised in sensory ganglia (e.g. substance P and CGRP) or the sympathetic chain (e.g. NPY). Recent studies point to the involvement of regulatory peptides in diseases of the gut and lung. These, together with detailed distribution studies, provide supportive data on the putative role of the peptides in the control of normal bowel and respiratory functions. The gastrointestinal and respiratory tracts were within the systems investigated by Feyrter during his original observations on the existence of specialised epithelial cells with a putative regulatory function (Feyrter, 1938). These "endocrine/paracrine" cells were found to be scattered in epithelial organs throughout the body. In fact, endocrine cells of the respiratory tract are frequently referred to as "Feyrter's cells". The term "regulatory peptides" was introduced as a generic term (Polak and Bloom, 1983) after the finding that active peptides are produced both by cells of the diffuse endocrine or APUD (amine precursor uptake and decarboxylation) system (Pearse, 1983) and autonomic/sensory nerves. These peptides are released into the circulation from endocrine cells or locally from nerve terminals or paracrine cells. The concept of "gut/brain" peptides was dispelled after the findings that the respiratory tract was provided abundantly with numerous active peptides produced by and released from mucosal endocrine cells and/or the innervation.

Animals

Immunoperoxidase labelling of albumin at the endothelial cell surface of frog mesenteric microvessels.

Albumin was visualised at the endothelial cell surface of perfused frog mesenteric microvessels using immuno-peroxidase labelling. Vessels in the mesenteries of pithed frogs were washed free of blood and then perfused with frog Ringer solutions containing bovine serum albumin (BSA) at a concentration of 20 mg BSA ml-1, followed by a brief Ringer flush to remove excess albumin from the vessel lumen. The tissues were fixed in 1% glutaraldehyde and a double antibody labelling technique used to identify albumin within the tissue. A dense layer of peroxidase reaction product was seen, which extended 25-50 nm into the vessel lumen. It appeared as a continuous layer lining the luminal openings of interendothelial cell clefts and vesicles open to the luminal cell surface. In some vessels a more irregular layer of peroxidase labelled albumin was seen extending 150 to 200 nm into the vessel lumen, whilst in others clumps of peroxidase labelled albumin were also seen within the vessel lumen. These data offer direct evidence that BSA does interact with the endothelial cell surface of perfused frog mesenteric microvessels but suggest that a proportion is loosley or non-specifically bound to the cell surface and can be removed by a brief Ringer flush. The remainder appears more tightly bound and resistant to Ringer flush.

Animals