Trigger point dry needling, manual therapy and exercise versus manual therapy and exercise for the management of Achilles tendinopathy: a feasibility study.

OBJECTIVES: The effects of trigger point dry needling (TDN) on myofascial trigger points (MTP) in Achilles tendinopathy (AT) are unknown. We conducted a study to test the feasibility of a large randomized controlled trial (RCT) to compare the effects of TDN to MT and exercise in a patient population with AT. METHODS: Twenty-two subjects were randomly assigned to a control (MT+Ex) or experimental group (TDN+MT+Ex) and completed eight treatment sessions over 4 weeks with follow up at 3 months. TDN was performed to MTPs in the gastrocnemius, soleus or tibialis posterior each session. The same MT and exercise program was conducted in both groups. RESULTS: Two of three criteria for feasibility were met. The attrition rate at 4-week and 3-month follow-up was 18.1% and 68%, respectively. Significant differences (p < .05) reported for within group analysis for FAAM, NPRS, pain pressure threshold and strength in both groups at 4 weeks and 3 months. The GROC was significant for MT + Ex at 3 months. No between group differences were found.  The MCID for the FAAM, GROC was surpassed in both groups at 4 weeks and 3 months and NPRS for the MT + Ex group at 4 weeks. DISCUSSION: A large RCT to investigate the effects of TDN on MTP in AT is not feasible without modifications due to low recruitment and high attrition rate. Modifications to study design should give consideration for closed or national health-care system for access to large patient populations and reduced financial burden to subjects. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03261504F.

Is there evidence to support an indication for surface replacement arthroplasty?: a systematic review.

AIMS: Surface replacement arthroplasty (SRA), compared with traditional total hip arthroplasty (THA), is more expensive and carries unique concern related to metal ions production and hypersensitivity. Additionally, SRA is a more demanding procedure with a decreased margin for error compared with THA. To justify its use, SRA must demonstrate comparable component survival and some clinical advantages. We therefore performed a systematic literature review to investigate the differences in complication rates, patient-reported outcomes, stress shielding, and hip biomechanics between SRA and THA. MATERIALS AND METHODS: A systematic review of the literature was completed using MEDLINE and EMBASE search engines. Inclusion criteria were level I to level III articles that reported clinical outcomes following primary SRA compared with THA. An initial search yielded 2503 potential articles for inclusion. Exclusion criteria included review articles, level IV or level V evidence, less than one year's follow-up, and previously reported data. In total, 27 articles with 4182 patients were available to analyze. RESULTS: Fracture and infection rates were similar between SRA and THA, while dislocation rates were lower in SRA compared with THA. SRA demonstrated equivalent patient-reported outcome scores with greater activity scores and a return to high-level activities compared with THA. SRA more reliably restored native hip joint biomechanics and decreased stress shielding of the proximal femur compared with THA. CONCLUSION: In young active men with osteoarthritis, there is evidence that SRA offers some potential advantages over THA, including: improved return to high level activities and sport, restoration of native hip biomechanics, and decreased proximal femoral stress shielding. Continued long-term follow up is required to assess ultimate survivorship of SRA.

Intra-operative digital imaging: assuring the alignment of components when undertaking total hip arthroplasty.

AIMS: The aims of this study were to examine the rate at which the positioning of the acetabular component, leg length discrepancy and femoral offset are outside an acceptable range in total hip arthroplasties (THAs) which either do or do not involve the use of intra-operative digital imaging. PATIENTS AND METHODS: A retrospective case-control study was undertaken with 50 patients before and 50 patients after the integration of an intra-operative digital imaging system in THA. The demographics of the two groups were comparable for body mass index, age, laterality and the indication for surgery. The digital imaging group had more men than the group without. Surgical data and radiographic parameters, including the inclination and anteversion of the acetabular component, leg length discrepancy, and the difference in femoral offset compared with the contralateral hip were collected and compared, as well as the incidence of altering the position of a component based on the intra-operative image. RESULTS: Digital imaging took a mean of five minutes (2.3 to 14.6) to perform. Intra-operative changes with the use of digital imaging were made for 43 patients (86%), most commonly to adjust leg length and femoral offset. There was a decrease in the incidence of outliers when using intra-operative imaging compared with not using it in regard to leg length discrepancy (20% versus 52%, p = 0.001) and femoral offset inequality (18% versus 44%, p = 0.004). There was also a difference in the incidence of outliers in acetabular inclination (0% versus 7%, p = 0.023) and version (0% versus 4%, p = 0.114) compared with historical results of a high-volume surgeon at the same centre. CONCLUSION: The use of intra-operative digital imaging in THA improves the accuracy of the positioning of the components at THA without adding a substantial amount of time to the operation. Cite this article: Bone Joint J 2018;100B(1 Supple A):36-43.

Diagnostic criteria of chronic inflammatory demyelinating polyneuropathy in diabetes mellitus.

OBJECTIVE: The possibility of co-association between diabetes mellitus (DM) and chronic inflammatory demyelinating polyneuropathy (CIDP) has long been a focus of interest as well as of clinical significance. As CIDP is a potentially treatable condition, it is diagnosis in the context of DM is of great importance. However, diagnostic criteria to identify CIDP in patients with diabetes are not available. We propose a diagnostic tool that should help clinicians to decide what is the probability that a patient with diabetes might have CIDP. METHODS: We list several clinical, electrophysiological, and laboratory parameters that, when combined, have the power of discriminating an immune-mediated neuropathy in patients with DM. By summing the points assigned to each of these parameters, we define four levels of probability for a patient with diabetes to have CIDP. To analyze the validity of the diagnostic toll, we applied it in three different patient populations: (i) Patients with diabetes with peripheral neuropathy, (ii) Patients with CIDP without DM, and (iii) Patients with diabetes with CIDP. RESULTS: The scores of patients with diabetes without CIDP ranged from -7 to 2, while those of patients with DM-CIDP ranged from 2 to 20. The scores of non-diabetic patients with CIDP were similar to those of patients with DM-CIDP and ranged from 6 to 16. The mean score of patients with DM-CIDP was 9.083, while the score of patients with CIDP was 11.16 and that of patients with diabetic polyneuropathy was -3.59. CONCLUSIONS: These results show that this diagnostic tool is able to identify patients with diabetes with overlapping CIDP.

Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.

Acetabular distraction: an alternative approach to pelvic discontinuity in failed total hip replacement.

A pelvic discontinuity occurs when the superior and inferior parts of the hemi-pelvis are no longer connected, which is difficult to manage when associated with a failed total hip replacement. Chronic pelvic discontinuity is found in 0.9% to 2.1% of hip revision cases with risk factors including severe pelvic bone loss, female gender, prior pelvic radiation and rheumatoid arthritis. Common treatment options include: pelvic plating with allograft, cage reconstruction, custom triflange implants, and porous tantalum implants with modular augments. The optimal technique is dependent upon the degree of the discontinuity, the amount of available bone stock and the likelihood of achieving stable healing between the two segments. A method of treating pelvic discontinuity using porous tantalum components with a distraction technique that achieves both initial stability and subsequent long-term biological fixation is described.

Efficacy of a static progressive stretch device as an adjunct to physical therapy in treating adhesive capsulitis of the shoulder: a prospective, randomised study.

BACKGROUND: Stress relaxation and static progressive stretch are techniques used for non-surgical restoration of shoulder range of motion for patients with adhesive capsulitis. OBJECTIVES: To compare a static progressive stretch device plus traditional therapy with traditional therapy alone for the treatment of adhesive capsulitis of the shoulder. DESIGN: Prospective, randomised controlled trial. PARTICIPANTS: Sixty patients with adhesive capsulitis of the shoulder were assigned at random to an experimental group or a control group. INTERVENTIONS: Both groups received three traditional therapy sessions per week for 4 weeks. In addition, the experimental group used a static progressive stretch device for 4 weeks. MAIN OUTCOME MEASURES: The primary outcome measure was shoulder range of motion (active and passive shoulder abduction, and passive shoulder external rotation). The secondary outcome measures were function [measured by the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire] and pain [measured using a visual analogue scale (VAS)]. RESULTS: At baseline, there were no differences between the two groups. However, after the intervention, there were significant (P<0.05) differences between the groups for all outcome parameters: 0.3 for mean VAS scores [95% confidence interval (CI) -0.6 to 1.1], -10.1 for DASH scores (95% CI -21.0 to 0.9), 21.2° for shoulder passive external rotation (95% CI 16.8 to 25.7), 26.4° for shoulder passive abduction (95% CI 17.4 to 35.3), and 27.7° for shoulder active abduction (95% CI 20.3 to 35.0). At 12-month follow-up, the differences between the groups were maintained and even increased for mean shoulder range of motion, VAS scores and DASH scores, with significant differences (P<0.001) between the groups: -2.0 for VAS scores (95% CI -2.9 to -1.2), -53.8 for DASH scores (95% CI -64.7 to -42.9), 47.9° for shoulder passive external rotation (95% CI 43.5 to 52.3), 44.9° for shoulder passive abduction (95% CI 36.0 to 53.8), and 94.3° for shoulder active abduction (95% CI 87.0 to 101.7). CONCLUSION: Use of a static progressive stretch device in combination with traditional therapy appears to have beneficial long-term effects on shoulder range of motion, pain and functional outcomes in patients with adhesive capsulitis of the shoulder. At 12-month follow-up, the experimental group had continued to improve, while the control group had relapsed.

The prevalence of cam-type femoroacetabular deformity in asymptomatic adults.

We performed a retrospective examination of the anteroposterior pelvic CT scout views of 419 randomly selected patients between April 2004 and August 2009 in order to determine the prevalence of cam-type femoroacetabular deformity in the asymptomatic population. The CT scans had all been undertaken for conditions unrelated to disorders of the hip. The frequency of cam-type femoroacetabular deformity was assessed by measuring the α-angle of each hip on the anteroposterior images. The α-angles were classified according to the Copenhagen Osteoarthritis Study. Among 215 male hips (108 patients) the mean α-angle was 59.12° (37.75° to 103.50°). Of these, a total of 30 hips (13.95%) were defined as pathological, 32 (14.88%) as borderline and 153 (71.16%) as normal. Among 540 female hips (272 patients) the mean α-angle was 45.47° (34.75° to 87.00°), with 30 hips (5.56%) defined as pathological, 33 (6.11%) as borderline and 477 (88.33%) as normal. It appears that the cam-type femoroacetabular deformity is not rare among the asymptomatic population. These anatomical abnormalities, as determined by an increased α-angle, appear to be twice as frequent in men as in women. Although an association between osteoarthritis and femoroacetabular impingement is believed to exist, a long-term epidemiological study is needed to determine the natural history of these anatomical abnormalities.

Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody.

The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.

Early implant failures in patients treated with Brånemark System titanium dental implants: a retrospective study.

Implant failure has been associated with factors such as poor bone quality, insufficient bone volume, implant instability, unfavorable implant loading, and smoking habits. Infections and host responses may also be important factors in dental implant failure. The objectives of the present study were to identify various explanatory factors associated with titanium implant failure. Forty subjects with stage 1 non-osseointegrated titanium dental implants (NOTI) ad modum Brånemark and 40 age- and gender-matched control subjects with successfully osseointegrated titanium implants (SOTI) were studied. Clinical data and gamma G immunoglobulin (IgG) antibody titers were studied. An independent t test revealed that significantly longer implants were placed in subjects with SOTI (P < .05). Statistically significant differences in bone shape and resorption (BSR) scores were found between SOTI and NOTI (P < .05). Logistic regression analysis identified 3 significant explanatory outcome variables: serum antibody avidity scores for Bacteroides forsythus (P < .0001), serum antibody titers to Staphylococcus aureus (P < .001), and the BSR scores (P < .05). Antibody avidity to B forsythus and antibody titer to S aureus were therefore the 2 most important factors associated with early implant failures and with a significant predictive ability. This indicates that immunologic factors are involved in osseointegration.

Synthesis and complexation properties of poly(ethylene glycol)-linked mono- and bis-dioxocyclams.

Both tri- and tetra(ethylene glycol) linked bis-chromium carbene complexes have been synthesized. These carbene complexes were photolyzed with N-protected imidazolines to give protected azapenams. These were transformed into polyether-linked basket dioxocyclams 4a,b and bis-dioxocyclams 5a,b. These compounds have cavities for the complexation of both "hard" and "soft" metal ions. The complexes of Ni, Ba, and Gd were synthesized.

Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates.

4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins. We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase.

Kinase-deficient neu proteins suppress epidermal growth factor receptor function and abolish cell transformation.

p185c-neu and epidermal growth factor receptor (EGFR) associate into an active heterodimer, and overexpression of these two receptors leads to a transformed phenotype. However, the association of EGFR and kinase-deficient Neu proteins (point mutant N757 or cytoplasmic domain deletion mutant N691stop) results in a defective or inactive heterodimeric complex. In this report we explore the biological consequences of heterodimerization between EGFR and wild-type (WT) or kinase-deficient mutant Neu proteins in living cells. We show that co-expression of EGFR and kinase-deficient Neu proteins abolished the synergistic transformation and tumorigenicity. Moreover, the normal responses of EGFR to ligand were significantly suppressed, e.g., loss of EGF-dependent transformation, reduced rate of receptor endocytosis and turnover, diminished DNA synthesis, and decreased EGF binding affinity. These results provide the first evidence that kinase-deficient Neu proteins suppress normal EGFR function and display a dominant negative mutant phenotype. Together with the stimulatory effects observed in cells forming active heterodimers, these studies provide a role for heterodimerization of EGFR and Neu/c-erbB2 in interreceptor activation and synergistic signaling which may be responsible for the transition from normal receptor function into oncogenesis.

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa.

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), gamma-melanocyte stimulating hormone (gamma-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the gamma-MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without 'spines' were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.

Demonstration by two-color flow cytometry that tyrosine kinase activity is required for down-modulation of the oncogenic neu receptor.

Expression of rat oncogenic neu receptor, p185T-neu (a growth factor receptor with constitutive tyrosine kinase activity), causes cells to become transformed. Treatment with anti-neu receptor monoclonal antibodies reverts the transformed phenotype by down-modulation of p185T-neu. Monoclonal antibody treatment of cells expressing normal neu receptor, p185C-neu (which lacks constitutive tyrosine kinase activity), does not result in down-modulation of p185C-neu. To understand further the role the biochemical activity of p185T-neu plays in transformation and endocytosis, we created a series of mutations in p185T-neu. We found that fibroblasts expressing the tyrosine kinase-defective mutants cannot form foci in culture, colonies in soft agar, or tumors in immunocompromised mice. To follow the antibody-induced endocytosis of neu receptors expressed in these transfectants, we developed a novel two-color flow cytometric assay and confirmed receptor localization by electron microscopy. Cells were treated with mAb7.16.4 over time. After 4 hr of antibody treatment, less than 50% of full-length p185T-neu and of mutant T691 remained on the cell surface, whereas internal expression of the neu receptors within these cells initially increased and then decreased to the original internal receptor level. In contrast, the level of kinase-deficient mutated neu receptors remaining on the cell surface initially decreased by 35%, but, after 4 hr of antibody treatment, the cell surface expression level returned to approximately the original level. Concurrently, fluctuations in expression levels were seen internally over time as well. These cell lines were also treated with gold-conjugated mAb7.16.4. Using electron microscopy, we consistently found the gold particles within multivesicular bodies of cell lines expressing full-length or mutated neu receptor. These data strongly suggest that the fate of the neu receptor, once internalized, is directed by its tyrosine kinase activity. When the kinase activity of the neu receptor is disrupted, the receptor is internalized but recycled to the cell surface, whereas neu receptors which have constitutive kinase activity are internalized and presumably degraded when engaged with anti-neu receptor mAb. Understanding the regulation of receptor endocytosis, degradation, and recycling will contribute to the development of novel therapeutic protocols to combat human malignancies, particularly those associated with the overexpression of the human homologue of the neu receptor, c-erbB2.

Hg-provocation of oral mucosa in patients with oral lichenoid lesions.

Five amalgam-bearing patients, with clinically and histologically confirmed oral lichenoid lesions, were tested by applying 0.5% Hg in petrolatum for 10 min to clinically normal mucosa. Control sites were exposed to petrolatum only. Four amalgam-bearing patients with no clinical evidence of oral lichenoid lesions served as controls; they were subjected to similar Hg and petrolatum exposure. After 24 h, biopsies were taken and immunocytochemically analyzed with monoclonal antibodies to lymphoid and nonlymphoid cells. No distinct differences could be detected between the Hg-exposed areas of the lichen patients and those of the nonlichen patients. Furthermore, normal mucosa exposed to petrolatum only showed a staining pattern in the lichen patients which was no different from the nonlichen patients. The findings are discussed with respect to possible mechanisms of development of lichen-like lesions in oral mucosa.

Analgesic efficacy of an ibuprofen-codeine combination in patients with pain after removal of lower third molars.

A double-blind, randomised analgesic trial was carried out in 165 patients undergoing surgical removal of one impacted lower wisdom tooth. In a two-dose regimen, the analgesic efficacy of the combination ibuprofen-codeine 200 mg : 30 mg was compared with that of acetylsalicylic acid-codeine 500 mg : 30 mg and codeine 30 mg. Each dose was taken when the patient needed pain relief. The intensity of the pain was measured on a visual analogue scale during the 10-h period after the first dose. The mean pain reduction by Dose 1 in patients on ibuprofen-codeine, acetylsalicylic acid-codeine and codeine was 64%, 45% and 26%, respectively, and the mean duration of effect was 8.3, 6.3 and 5.6 h. According to the pain reduction, duration of effect and pain reduction index after Doses 1 and 1 + 2, there was a significant difference between ibuprofen-codeine and the other two drugs. The maximum pain reduction within 4 hours was 84% with ibuprofen-codeine. This was significantly different from the reduction achieved both with acetylsalicylic acid-codeine (64%) and codeine (35%). Seventeen patients reported adverse events: 5 on ibuprofen-codeine, 4 on acetylsalicylic acid-codeine and 8 on codeine. The most common events were tiredness and vertigo. It is concluded that the combination ibuprofen-codeine 200 mg : 30 mg had greater analgesic efficacy compared to the combination acetylsalicylic acid-codeine 500 mg : 30 or codeine 30 mg in patients with pain after removal of the lower third molars.

Expression of c-erbB-2 in in situ and in adjacent invasive ductal adenocarcinomas of the female breast.

Several lines of evidence have demonstrated that expression of the c-erbB-2 gene product contributes to the malignant phenotype. We and others have determined that c-erbB-2 is substantially expressed in most ductal in situ carcinomas of the comedo type, but not in other patterns of ductal carcinoma in situ or in atypical ductal hyperplasia of the breast. In the present investigation, by immunohistochemistry we inquired whether invasive ductal adenocarcinomas retained the c-erbB-2 expression status of the in situ carcinomas from which they derived. Of twelve specimens containing both cribriform/micropapillary in situ and derivative invasive adenocarcinomas in the same section, all tumor cells were negative for c-erbB-2 expression. In thirteen in situ carcinomas of the comedo type, with identifiable invasive components, ten had definite c-erbB-2 expression, and in every case there was comparable c-erbB-2 protein staining of in situ and invasive components; in three of these ten cases the staining in the in situ component tended to be more intense. These findings imply that a significant proportion of invasive mammary adenocarcinomas expressing c-erbB-2 protein is derived from ductal in situ carcinomas of the comedo type.

The microspore-derived embryo ofBrassica napus L. as a tool for studying embryo-specific lipid biogenesis and regulation of oil quality.

A time-course study of lipid accumulation in microspore-derived embryos and developing zygotic embryos of rapeseed (Brassica napus L. ssp.oleifera) is presented. Rapid storage fat (triacylglycerol) biosynthesis was induced in microspore-derived embryos of oilseed rape (cv 'Topas') when the embryos were transferred from standing cultures (10 ml) to fresh medium (75 ml) and shake cultured. Triacylglycerols accumulated, after a lag period of 7 days, at a linear rate of approximately twice that of the developing zygotic embryo. The fatty acid composition of triacylglycerols in microspore-derived embryos closely parallelled that of the developing zygotic embryos. In the microspore-derived embryos, the amount of phosphatidylcholine, the major substrate for the production of polyunsaturated fatty acids in oilseeds, remained constant during the linear phase of triacylglycerol production, whereas it increased steadily in the zygotic embryos. The fatty acid composition of individual cotyledons from microspore embryos shake cultured for 15 days was compared with that of individual mature seeds. Relative amounts of the major fatty acids, i.e. palmitic, oleic and linoleic acids, were essentially the same, whereas the microspore-derived embryos had about 35% less stearic acid and 35% more linolenic acid than the mature seeds. Variation in the amounts of oleic, linoleic and linolenic acids between seeds was similar to that found between cotyledons of microspore-derived embryos, whereas variation in palmitic and stearic acid levels was significantly lower between microsporederived cotyledons than between the seeds. The results indicate that microspore-derived embryos from shake cultures should be convenient for use in studying the regulation of oil biosynthesis and for rapidly screening for oil quality in genetically altered rapeseed.

Analysis of c-erbB-2 protein expression in conjunction with DNA content using multiparameter flow cytometry.

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c-erbB-2 protooncogene is amplified in 10-40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c-erbB-1 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c-erbB-2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c-erbB-2 expression in populations of ethanol-fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c-erbB-2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.