Surgical repair of a meningoencephalocoele in a kakapo (Strigops habroptilus).

CASE HISTORY: A kakapo (Strigops habroptilus) chick hatched on an off-shore island of New Zealand with a small white mass protruding through the cranial skin of the head. The chick's growth followed a normal pattern for kakapo but at 3 weeks of age the cranium mass was non-reducible and fixed in place and the chick was removed from the island for diagnostic imaging and hand-rearing. CLINICAL FINDINGS AND TREATMENT: A computed tomography (CT) examination revealed a full-thickness circular defect in the central cranium with suspected herniation of brain and dura. Surgery was performed at 37 days of age, and the herniated dura was dissected from the open fontanelle. Attempts to reduce the herniated tissue were unsuccessful, so the herniated dura and cortex were clamped and resected. The dura was closed and the periosteum of the skull was scarified and monofilament polypropylene mesh was secured tautly over the fontanelle. The mesh graft was infused with autologous bone marrow harvested from the ulna in an attempt to stimulate osteogenesis in the mesh repair. The skin flap was then closed. Post-operative recovery and healing were without complication. A CT examination 4 weeks after surgery showed no recurrence of the hernia, and a composite of mesh and scar over the open fontanelle which had reduced in diameter. The chick was released back onto an off-shore island with a radio transmitter and it continues to be monitored regularly. PATHOLOGICAL FINDINGS: The tissue resected at surgery consisted of a cylindrical core of cerebral parenchyma overlain by a mildly hyperplastic epidermis, and large amounts of oedematous fibrovascular tissue arising from the leptomeninges. DIAGNOSIS: Rostral parietal meningoencephalocoele. CLINICAL RELEVANCE: This is the first report of successful surgical resolution of a meningoencephalocoele in any bird. Techniques from human neurosurgery were adapted for the unique anatomical features of the avian skull. The risks of the procedure included increased intra-cranial pressure resulting in anaesthetic complications or death, cerebrospinal fluid leakage, meningitis or recurrence of the meningoencephalocoele. In the longer term, there was a risk of developmental deficits in cognition or behaviour. None of these complications eventuated in the short to medium term, probably due to the small size of the meningoencephalocoele.

Concentrations of calcium and 25-hydroxycholecalciferol (vitamin D3) in plasma of wild kakapo (Strigops habroptilus) living on two islands in New Zealand.

AIMS This preliminary study had the objectives of describing the concentrations of ionised calcium and 25-hydroxycholecalciferol (25(OH)D3) in the blood of wild kakapo (Strigops habroptilus) living on two islands in New Zealand, and to determine the effects of supplementary feeding on these blood parameters. METHODS Blood samples were obtained from 33 kakapo living on two offshore islands during routine health checks in 2015. Birds on Hauturu were sampled in May (n=5) and birds on Whenua Hou were sampled in July (n=15) and November (n=26). Of the birds sampled on Whenua Hou in November, 15 received supplementary food prior to sampling. Samples were analysed for pH, and concentrations of ionised calcium, total calcium, phosphorous, total protein, albumin, globulin, uric acid and 25(OH)D3. RESULTS Concentrations of ionised calcium did not differ between unsupplemented birds on the two islands, nor between supplemented (median 1.17 (95% CI=1.12-1.20) mmol/L) and unsupplemented (median 1.09 (95% CI=1.08-1.14) mmol/L) birds sampled in November on Whenua Hou (p>0.05), and were comparable with published normal ranges for other psittacines. Concentrations of 25(OH)D3 did not differ between unsupplemented birds on the two islands (p>0.05), but were higher in supplemented (median 8.00 (95% CI=4.76-8.45) nmol/L) than unsupplemented (median 0.00 (95% CI=-0.16-0.48) nmol/L) birds on Whenua Hou (p<0.001). All values were much lower than published ranges for healthy psittacines. There was no difference between male and female birds on Whenua Hou for any parameter measured (p>0.05). CONCLUSIONS AND CLINICAL RELEVANCE The calcium status of the kakapo in this study was comparable to other wild psittacines, however concentrations of 25(OH)D3 were much lower. The concentrations of 25(OH)D3 may be within the normal range for the species, however further data are required to confirm this. The significant increase in concentrations of 25(OH)D3 in supplementary fed birds suggests that this food was providing more of the nutrient than the wild diet at that time of year, although the effects of this are unknown. Further investigation is required into the calcium and vitamin D3 status of kakapo, across a wider range of locations, seasons and ages. This would help define normal ranges for these parameters, allow interpretation in clinically abnormal individuals, and guide the refinement of supplementary foods. This information would, therefore, assist the future conservation management of this critically endangered species.

Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand.

CASE HISTORY: Health monitoring of tuatara (Sphenodon punctatus) at Auckland Zoo between 2001 and 2009 showed that 58/93 tuatara had been affected by dermatitis of unknown origin. From 2011 onwards, cases of suspected fungal dermatitis underwent extensive diagnostic investigations. CLINCAL FINDINGS: Six cases of dermatomycosis were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five tuatara. LABORATORY FINDINGS: Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA. DIAGNOSIS: All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates. CLINICAL RELEVANCE: These are the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Tuatara at the Auckland Zoo are now routinely examined every 3 months and tissue samples from any lesions sent for histopathology and fungal culture. Further work to elucidate the epidemiology and significance of P. australasiensis infections in reptiles in New Zealand is important for both welfare and conservation purposes.

Lipid infusion in the management of poisoning: a report of 6 canine cases.

Intravenous administration of lipid is a relatively new treatment in the management of toxicity from lipophilic compounds. It is used in human medicine in the treatment of toxicity from lipophilic local anaesthetics and cardiotoxic drugs and can result in dramatic improvement in clinical status. We present six cases of poisoning in dogs successfully treated with lipid infusion after ingestion of ivermectin (3), moxidectin (2) and baclofen (1). The dogs ranged in age from eight weeks to 14 years, and weighed 4-30 kg. Intravenous lipid therapy was started between six and eight hours and 22 hours after ingestion, and all the dogs responded well. In four dogs, there was clinical improvement within one hour; one had improved within two hours and the other within 4.5 hours of lipid administration. The only adverse effect of lipid infusion reported was mild swelling and pain after extravasation in one case which resolved with conservative management. All the dogs were discharged within 24-52 hours after exposure (7-46 hours after the start of lipid administration), and none developed any apparent sequelae.

Interpretation criteria in Western blot diagnosis of Lyme borreliosis.

This study reviews the Lyme borreliosis Western blot interpretation process, including what bands are classed as specific, the number of bands needed for a positive result, the role of band intensity and the use of clinical information. In 2008, 3688 patients (4223 serum samples) were tested by enzyme immunoassay (EIA), with 832 patients tested by confirmatory in-house IgG Western blot: 272 patients were Western blot-positive, 170 were weak positive, 156 were equivocal and 234 were negative. These results were assessed, and a review of interpretation criteria from both the USA and Europe was carried out. New interpretation criteria and a testing algorithm were developed. The revised criteria changed the results in 109/3688 (3%) patients and produced significantly more Western blot-positive and weak-positive patients than with the current criteria (485 vs. 442, P < 0.0001). In total, 76 patients who were negative/equivocal became positive, which may have led to a change in their management. Conversely, 33 patients who were weak-positive became equivocal but their management may not have been affected. The authors believe that the revised criteria have simplified blot interpretation and improved the sensitivity and robustness of their Western blot method. Using a protocol tailored to patients that incorporates clinical characteristics means that the entire process will be easier and will aid the management of patients.

Pineocytoma in a lowland anoa (Bubalus depressicornis).

This report describes the first case of a pineocytoma in an 18-year-old female lowland anoa (Bubalus depressicornis). The tumour grossly appeared as a focal, non-infiltrative, yellow-tan, encapsulated mass occupying the normal anatomical location of the pineal gland. Microscopical, immunohistochemical and electron microscopical findings were consistent with a diagnosis of pineocytoma an entity not previously described in this member of the buffalo subgenus species.

Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves western blot sensitivity.

AIMS: This study evaluates the use of local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen for in-house IgG western blots in the routine diagnostic setting by comparing it with the current in-house protocol. METHODS: Sera from 233 patients from areas of Scotland with low and high prevalence for Lyme borreliosis were tested by western blots prepared from reference strain antigen (B burgdorferi sensu stricto) and mixed antigen (B burgdorferi sensu stricto and B afzelii). Results were scored using original and revised criteria, and results were compared. RESULTS: The mixed antigen produced significantly more bands than the reference antigen. Using the original interpretation criteria the mixed antigen produced more positive results than the reference antigen (90 versus 85). When the revised criteria were applied to the mixed antigen there were 14 more patients with positive results than with the reference antigen (99 versus 85); this difference was significant. Although 22 patients were positive with the mixed antigen and revised criteria, but negative/equivocal with the reference antigen, eight patients who were positive with reference antigen remained negative with the mixed antigen. The positive predictive value of the two antigen preparations was the same (96%). The negative predictive value of the mixed antigen with revised criteria was higher than the reference antigen (96% versus 88%), but the specificity was similar (97% versus 98%). CONCLUSIONS: The mixed antigen and revised interpretation criteria have been successfully incorporated into the routine diagnostic testing service, increasing the sensitivity of the in-house IgG western blot test for Scottish patients.

Development of real time PCR to detect Toxoplasma gondii and Borrelia burgdorferi infections in postal samples.

BACKGROUND: Most of the samples sent to reference laboratories are delivered by post. Thus, diagnostic PCR tests on blood samples have to be performed using methods which are optimised and validated for such conditions. There is a low probability that the organisms Toxoplasma gondii and Borrelia burgdorferi will be present. AIM: To confirm that robotic extraction methods followed by real time PCR will detect as little as one organism/test sample in postal specimens. METHODS: Human blood samples spiked with decreasing numbers of each organism (range 10(5)-1/per extract) were extracted using two commercial kits on a Qiagen BioRobot EZ1 Workstation. Extracts of whole blood and blood fractions were tested by real time PCR. The effect of storage of blood for 1-6 days at room temperature was also investigated. RESULTS: Maximum sensitivity (1 organism/test sample) was achieved for T gondii with either extraction method; the sensitivity for B burgdorferi was between 1 and 10 organisms/test. Whole blood was the most suitable sample to extract, as both organisms were as likely to be detectable in the red cell as the white cell fraction. Sensitivity was not reduced by storing spiked samples at room temperature for up to 6 days. Inhibitory effects on PCR were not a significant problem provided that samples were extracted using the blood extraction kit. CONCLUSIONS: Using appropriate robotic extraction methods, both T gondii and B burgdorferi can be detected by real time PCR with near maximum possible sensitivity in whole blood samples. Blood samples can be transferred to reference laboratories by post without loss of sensitivity over the likely transit period.

Polycyclic aromatic hydrocarbon-degrading Mycobacterium isolates: their association with plant roots.

Five environmental mycobacterium isolates that degrade polycyclic aromatic hydrocarbons (PAHs) were associated with barley root surfaces after growth of the seedlings from inoculated seed. Mycobacterium cells were detected along the total root length for four of these isolates. These PAH-degrading mycobacterium strains had hydrophilic cell surfaces, whereas one strain, MCS, that was hydrophobic had reduced association along the root length with no cells being detected from the root tips. The root-tip-competent strain, KMS, was competitive for its root association in the presence of the root-colonizing pseudomonad, Pseudomonas putida KT2440. All mycobacterium strains utilized simple sugars (fructose, glucose) and the trisaccharide 6-kestose, present in barley root washes, for planktonic growth, but they differed in their potential for biofilm formation under in vitro conditions. Mineralization of pyrene by the KMS strain occurred when the components in the barley root wash were amended with labeled pyrene suggesting to us that mineralization could occur in plant rhizospheres containing such mycobacterium strains.

The use of local isolates in Western blots improves serological diagnosis of Lyme disease in Scotland.

Nine Scottish Borrelia burgdorferi isolates were investigated in IgG Western blot tests. Sera previously found to be positive and negative when tested by routine Western blots prepared from reference strain B. burgdorferi sensu stricto antigen had different outcomes with these isolates. Two isolates, E5 (Borrelia afzelii) and G4 (B. burgdorferi sensu stricto) performed well, reproducing Western blot-positive results in 90 and 95% of tests, respectively. When antigens from both isolates were incorporated into a single IgG Western blot, the results of a panel of sera were improved when compared to the routine reference strain IgG Western blot. All of the sera positive by the routine Western blot remained positive using the Scottish isolate antigen mix. Twenty-three of the 25 negative sera remained negative and two produced an equivocal result. Of the 15 samples that tested IgG Western blot equivocal with the B. burgdorferi sensu stricto reference strain, 11 (73%) became weak or strong positive when tested with the B. afzelii/B. burgdorferi sensu stricto antigen mix (chi(2)=14.35, Yates' correction, P<0.001). In seven of these, a clinical picture of Lyme disease was consistent with the new results. The use of Scottish strains of B. afzelii and B. burgdorferi sensu stricto to provide antigen for the IgG Western blot improves the diagnosis of Lyme disease for patients in Scotland.

Strategies for detecting toxoplasma immunity.

A strategy for identifying toxoplasma immunity in pregnancy must provide good evidence of immunity but not falsely reassure; that for immunocompromised patients should identify immunity and also the risk of reactivated toxoplasmosis. Using sera from both of these patient groups, the performance of an in-house IgG EIA and two commonly used commercial assays (Abbott AxSYM Toxo-G and Eiken latex test) were compared with the dye test. False-positive results were obtained using the IgG enzyme immunoassay (EIA) and AxSYM Toxo-G, and false negatives using all three screen tests. During pregnancy, positive results may falsely reassure, and patients should be tested for toxoplasma-specific IgM to differentiate between current infection and immunity. In immunocompromised patients, positive results indicate immunity but negative results do not exclude it; these should be tested by dye test. Despite these reservations, we have demonstrated that immunity screening can be performed within a district general hospital.

Toxoplasma gondii from liquid nitrogen for continuous cell culture: methods to maximise efficient retrieval.

This study aims to increase the efficiency of continuous growth of Toxoplasma gondii in HeLa cells from tachyzoite stocks frozen in liquid nitrogen. Freezing and retrieval of tachyzoites for continuous cell culture requires more stringent protocols than those published for animal culture. The freezing and retrieval conditions are optimised so that a quality harvest (> or = 1 x 10(6) tachyzoites/mL, > or = 90% viability) can be produced using T. gondii recovered from liquid nitrogen as fast and reliably as possible. Retrieval success rate increased from 36% to 100%. An improved freezing procedure using chilled reagents and freshly harvested parasites, and adoption of an effective recovery protocol with retrieval of 3 x 10(7) tachyzoites into 75 cm2 flasks, change of maintenance media after six hours and subsequent blind passage all contributed to this success. The result is faster and more dependable production of T. gondii for diagnostic and experimental use.

How to detect current toxoplasma infection.

This study seeks to identify the best way to detect current toxoplasma infection for district general hospital laboratories. One hundred 'ordinary' and 174 'difficult' sera are categorised into either an 'evidence' or 'no evidence' group for current toxoplasma infection. Twelve test protocols are investigated using different combinations of one whole antibody latex test (Eiken Toxoreagent), one in-house specific IgG enzyme-linked immunosorbent assay (ELISA) and three specific IgM assays (Toxo-ISAGA, in-house BAM ELISA IgM and Toxonostika ELISA M). The Eiken latex and in-house IgG assays produced significantly fewer false-negative results than were obtained with the single IgM test or the IgG and IgM test protocols (P<0.05), but a greater number of false-positive results (102/274 and 115/274, respectively). Of the IgM assay test protocols, the three IgM assays in combination produced the least number of false-negative results (1/274). However, a significantly greater number of false-positive results were produced than with one or two IgM tests or an IgG and any IgM test in combination (P<0.001). We recommend testing with three IgM tests, or a whole antibody (Eiken) or IgG-specific assay, and that positive or clinically important negative samples be referred to a reference laboratory for confirmation.

Success in the toxoplasma dye test.

OBJECTIVES: To compare the success rate of the toxoplasma dye test using different accessory factors (human serum as a source of complement) and different batches of tachyzoites produced in vivo and in vitro. METHODS: Twenty-five accessory factors were used in the dye test to assess both types of tachyzoite. Batches of tachyzoites were produced in vivo (n = 49) and in vitro (n = 23) and their performance assessed against panels of accessory factors. Performance was recorded as success or failure (incorrect results, total killing or no killing). RESULTS: With in vivo tachyzoites 21/25 accessory factors were successful in P > or = 1 dye test runs, whereas with in vitro tachyzoites all 25 were successful. One or more failure was recorded for 19/25 and 12/25 accessory factors using in vivo and in vitro tachyzoites, respectively (P < 0.05). The number of successful dye test runs was less for in vivo (92/141, 65%) than in vitro (140/163, 86%) tachyzoites (P < 0.001). This was due to a higher success rate in citrated accessory factors used for in vitro tachyzoites compared to the corresponding uncitrated accessory factors used for in vivo tachyzoites (P < 0.001). Success in the dye test was recorded for 48/49 and 23/23 batches of in vivo and in vitro tachyzoites, respectively. The number of successful dye test runs was lower with in vivo (156/234, 67%) than in vitro (116/142, 82%) tachyzoites (P < 0.01). CONCLUSIONS: Success in the dye test may be due to the accessory factor, tachyzoites, or a combination of both. Problems due to the accessory factor can be minimized by careful quality control or use of modification procedures. Tachyzoites produced in vitro may also increase success in the dye test. Careful selection of accessory factor/tachyzoite combination makes it possible to use the dye test in a district general hospital.

Toxoplasma dye test using cell culture derived tachyzoites.

AIMS: To assess the diagnostic usefulness of Toxoplasma gondii tachyzoites produced by serial passage in HeLa cell culture. METHODS: Tachyzoites derived from serial passage in cell culture were used in the dye test. Human sera were also examined to determine their suitability for use as accessory factor. Using the optimum conditions, the dye test using cell culture derived tachyzoites was compared with the current method of production (animal culture) on 105 randomly selected sera. Start up and maintenance costs of each system were compared. RESULTS: Tachyzoites in most cell culture harvests (84%) from both early and later passes were useable. Tachyzoite yield and viability were maintained during serial passage in cell culture. Sodium citrate was used to modify accessory factor and improve its suitability. The performance of the accessory factor was improved by the addition of 1% and 3% sodium citrate for the current and cell culture systems, respectively. Under optimum conditions, dye test titres using cell culture and current systems were compared on 105 randomly selected sera. The results from 92 of 105 (87.6%) patients agreed or were within one dilution, but all discrepancies were resolved on re-testing. Start up costs for the current system would be 2.5 times and overall maintenance three times more expensive than cell culture. CONCLUSIONS: Tachyzoites derived from cell culture can be used routinely in the dye test. Production in cell culture is more cost effective than animal culture. It is possible for general hospitals to perform the dye test, thus obtaining faster and more specific results.

Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex.

The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates. The LDLC gene encodes ldlCp, an approximately 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus. Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells. The corresponding mRNA was not detected in ldlB mutants. LDLB encodes an approximately 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs. Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially full-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae. We have found that in wild-type cell cytosols, ldlCp is a component of an approximately 950-kDa "ldlCp complex," which is smaller, approximately 700 kDa, in ldlB cytosols. Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.

Cell-culture system for continuous production of Toxoplasma gondii tachyzoites.

The aim of this study was to identify a sustainable cell line and culture method that could continuously provide a sufficient quantity of Toxoplasma gondii tachyzoites to serve the needs of a general hospital laboratory. Three continuous cell lines (HeLa, LLC and Vero) and three cell-culture methods (culture in conventional flasks, culture in membrane-based flasks and an automated culture system) were investigated. In multiplicity-of-infection and time-course experiments, HeLa was the cell line of choice. Harvests from HeLa cells had significantly higher tachyzoite yields than those from LLC cells (P<0.00005) or Vero cells (P<0.05). Membrane-based flasks gave higher yields (6.15x10(6) tachyzoites/ml) than conventional flasks (1-2x10(6) tachyzoites/ml) initially, but these were not sustained. The automated cell-culture system was unsuitable for parasite culture. Continuous passage in 25 cm2 flasks was successful, yielding 1x10(6) tachyzoites/ml; viability exceeded 90% after 96-120 h of infection throughout 38 passes, during which time the viability improved and the time to harvest became more consistent. Toxoplasma gondii grown in continuous culture in HeLa cells can provide a regular supply of viable tachyzoites. Demonstration that HeLa-derived tachyzoites could be used for the dye test confirms the potential of this in vitro system for use in general hospital laboratories.

Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain.

Structures of secreted forms of the human type I and II class A macrophage scavenger receptors were studied using biochemical and biophysical methods. Proteolytic analysis was used to determine the intramolecular disulfide bonds in the type I-specific scavenger receptor cysteine-rich (SRCR) domain: Cys2-Cys7, Cys3-Cys8, and Cys5-Cys6. This pattern is likely to be shared by the highly homologous domains in the many other members of the SRCR domain superfamily. Electron microscopy using rotary shadowing and negative staining showed that the type I and II receptors are extended molecules whose contour lengths are approximately 440 A. They comprised two adjacent fibrous segments, an alpha-helical coiled-coil ( approximately 230 A, including a contribution from the N-terminal spacer domain) and a collagenous triple helix ( approximately 210 A). The type I molecules also contained a C-terminal globular structure ( approximately 58 x 76 A) composed of three SRCR domains. The fibrous domains were joined by an extremely flexible hinge. The angle between these domains varied from 0 to 180 degrees and depended on the conditions of sample preparation. Unexpectedly, at physiologic pH, the prevalent angle seen using rotary shadowing was 0 degrees , resulting in a structure that is significantly more compact than previously suggested. The apparent juxtaposition of the fibrous domains at neutral pH provides a framework for future structure-function studies of these unusual multiligand receptors.

Immunoelectron microscopy of low density lipoproteins yields a ribbon and bow model for the conformation of apolipoprotein B on the lipoprotein surface.

In the present study, the relative positions of 11 anti-apolipoprotein B monoclonal antibodies have been mapped onto the surface of human low density lipoproteins by electron microscopy. As the epitopes recognized by these antibodies have been previously located on the primary sequence of apoB, these data provide a map of the configuration of the protein on the surface of the LDL. The first 89% of apoB-100 may be modeled as a thick ribbon that wraps once around the LDL, completing the encirclement by about amino acid residue 4050. The thickness of the ribbon is sufficient to penetrate the monolayer, so that it makes contact with the core. There is a kink in the ribbon beginning almost halfway along its length at approximately apoB-48. The C-terminal 11% of apoB constitutes the "bow," an elongated structure of about 480 residues, beginning at 4050 and stretching back into one hemisphere and then crossing the ribbon into the other hemisphere between residues 3000 to 3500, thus bringing sequences in the C-terminal portion of apoB-100 near to the suggested binding site for the LDL receptor. The C-terminal sequences may act as a negative regulator of LDL receptor binding, in agreement with Parhofer et al, 1992. J. Clin. Invest. 89: 1931-1937, who reported the enhanced clearance from plasma of apoB-89-containing lipoproteins. It is proposed that in VLDL the bow could function to inhibit binding to the receptor; during lipolysis to form LDL, it is suggested that these C-terminal inhibitory sequences forming the bow would move sufficiently to allow interaction with the LDL-receptor.

Identification of apolipoprotein B100 polymorphisms that affect low-density lipoprotein metabolism: description of a new approach involving monoclonal antibodies and dynamic light scattering.

Rare mutations in apolipoprotein B (apoB) can cause defective binding of low-density lipoproteins (LDLs) to the LDL receptor, leading to elevated plasma cholesterol levels and premature atherosclerosis. This communication describes a novel approach to study the effects of apoB mutations on LDL metabolism. Monoclonal antibody MB19 identifies a common polymorphism in apoB, an Ile/Thr substitution at residue 71, by binding with a 60-fold higher affinity to apoB(Ile71)-containing LDL. Because each LDL contains a single apoB, a maximum of two LDLs may be bound by the bivalent monoclonal antibody. Thus, at the appropriate concentration, an equivalent amount of MB19 will promote substantial dimer formation of LDL containing the strongly binding apoB(Ile71), but little dimer formation of LDL containing the weakly binding apoB(Thr71). For LDL isolated from heterozygous individuals, the amount of dimer formed, determined by dynamic light scattering, yields an estimate of the allelic ratio of the two forms of LDL. For such individuals, not only the effect of the polymorphism recognized by MB19 but also the effects of other polymorphisms on the LDL allelic ratio can be determined. Examination of six normolipemic MB19 heterozygotes gave percent allelic ratios between 48:52 and 51:49 tight:weak-binding LDL, not significantly different from a 50:50 ratio. These individuals were also heterozygous for six common apoB polymorphisms, allowing calculation of the odds that each of these polymorphisms caused significant alterations in lipid levels. In contrast, the rare mutation at residue 3500 causing defective binding to the LDL receptor and familial defective apoB100 (FDB) resulted in substantial changes (26:74 and 13:87) in LDL allelic ratio in both of two FDB individuals examined.