Site-directed mutagenesis of the N-linked glycosylation site in platelet-derived growth factor B-chain results in diminished intracellular retention.

Pulse-chase analysis of human platelet-derived growth factor (PDGF) B-chain was conducted in stably transfected Chinese hamster ovary cells to determine precisely the kinetics of processing, intracellular trafficking and secretion. Newly synthesized 31 kDa monomers of the B-chain (p31) dimerized rapidly via disulfide bonds to a p54 species (t1/2 < 30 min). The p54 dimer was processed to a group of intracellular, cell surface (suramin-releasable) and secreted forms whose rates of appearance and disappearance from the cell were measured over a 48 h period. The newly synthesized p31 species was quantitatively converted to p27 by treatment with endoglycosidase H, consistent with efficient N-glycosylation at a site in the N-terminal propeptide region (Asn63-Met64-Thr65). Interruption of B-chain glycosylation by oligodeoxynucleotide-directed mutagenesis resulted in a significant increase in suramin-releasable forms at the cell surface (p34-38) and a concomitant decrease in accumulation of an intracellular p24 species. The glycosylation-defective mutant exhibited slight increases in receptor binding and mitogenic activity. Our results suggest that N-linked glycosylation of the B-chain is not important for formation of mitogenically active protein, but that it plays a role in early intracellular sorting and proteolytic processing events.

Efficient transformation of Serratia marcescens with pBR322 plasmid DNA.

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.

Medial calcification (whitlockite) in the aorta.

Calcified deposits in the tunica media of the human aorta have been studied in 128 cases by light microscopy and by electron microscopy and analytical methods in selected samples. Although dissolved and not visible in routine histology with alum hematoxylin stains, such calcification can be clearly seen after methylene blue staining in the form of unstained refractile particles of 1-2 microns size. These are found between the elastic laminae chiefly in the inner two-thirds of the media and appear at about age 20. By X-ray diffraction supported by energy dispersive X-ray analysis, they have been identified as whitlockite (Ca,Mg)3(PO4)2. Statistical analysis shows a significant increase in numbers with age and significant differences in severity related to county of origin but no differences between sexes or races and no correlation with deaths related to cardiovascular diseases. Among various substructures of the aortic wall, no unique crystal precursor was identified. Possible etiologic factors and clinicopathologic significance are considered.

Mediastinal histoplasmosis with abscess.

A mediastinal mass of clinically undetermined nature was found at autopsy to be an enormous abcess due to secondary infection in granulamatous caseating mediastinal nodes caused by Hitosplasma capsulatum. Other complications of mediastinal histoplasmosis are briefly reviewed.

The black thyroid associated with minocycline therapy. A local manifestation of a drug-induced lysosome/substrate disorder.

Two cases of black discoloration of the thyroid are reported, each in a young woman treated for acne; the first with minocycline alone, the second with tetracycline and minocycline. Light microscopy showed numerous brown granules in the cytoplasm of the follicular epithelium, with large brown deposits in the colloid, including, in Case 2, numerous crystalline forms. The association between drug therapy and pigment accumulation (in conjunction with animal experiments) was taken to indicate some form of causal relationship, and minocycline residues were anticipated. Histochemical studies indicated that the granules were lipofuscin and electron microscopy showed an increase in phagolysosomes but no novel structures. There was no evidence for persisting minocycline, or its derivatives, although these could not be excluded. The black thyroid appears to be a disorder at lysosome/substrate level, induced by drug action, particularly by minocycline, and manifested by an accumulation of heterogeneous intracellular material with the histochemical and electron microscopic appearances of lipofuscin.

Calcium oxalate in sarcoid granulomas. With particular reference to the small ovoid body and a note on the finding of dolomite.

The nature, prevalence, and specificity of birefringent calcific particles in granulomas of sarcoidosis have been examined, including histochemical reactions, single particle, and microchemical analyses. Particular attention was paid to small ovoid forms of which most were calcium oxalate monohydrate. Larger crystals, those within giant cells, and the birefringent component of a Schaumann complex were also calcium oxalate. Small ovoids appeared to originate in macrophages and to be precursors of other forms; they were found in 86% of lymph nodes and 73% of surgical lung specimens. They were not specific for sarcoidosis. Organisms could not be certainly identified in them. Their origin is discussed in relation to activated macrophages, calcium, and oxalate metabolism, and the role of calcium oxalate in granulomas is considered. Four particles from two cases were dolomite and two were a calcium-sulphur compound. The biologic origin of dolomite is reviewed.

Carcinoma of the colon with undifferentiated, carcinoid, and squamous cell features.

An undifferentiated small cell tumor of the colon that was found by light- and electron-microscopic study to possess features of a malignant carcinoid merged into keratinizing squamous cell carcinoma. Single cells contained both dense-core secretory granules and tonofilaments. The implications of this combination in relation to theories of tumor origin and differentiation are briefly discussed.

Calcium oxalate crystals in the thyroid. Their identification, prevalence, origin, and possible significance.

Calcium oxalate crystals are not encountered in normal animal tissues, except for the human thyroid, where they were found in 79 of 100 routine consecutive autopsies. They appear during childhood, and numbers of crystals increase with age. In diffuse hyperplasia, prevalence was higher, but crystals were fewer than expected. In adenomas and carcinomas, crystals were decreased except for three cases with a striking focal increase. None was found in 22 adult primate thyroids. After Clorox digestion of human thyroids, calcium oxalate dihydrate was identified by x-ray diffraction and infrared spectroscopy. Origin, tissue and species localization are discussed in relation to ascorbate metabolism, thyroperoxidase, and calcitonin. Possible metabolic roles are suggested. Calcium oxalate crystals injected in animals and humans initiate a foreign body reaction with giant cells. In Hashimoto's thyroiditis, crystals disappear but occasionally remain with giant cell reaction. In subacute thyroiditis, granulomas are related more to colloid than to crystals.

Endometriosis of the colon with mixed "germ cell" tumor.

A 42-year-old woman was found to have endometriosis of the oviducts. Five years later she had a hysterectomy and oophorectomy, the abnormal findings at this time being endometriosis with a chocolate cyst of one ovary. At a third operation, seven years after the first, she was found to have endometriosis of the colon, associated with malignant tumor in the serosa of the intestine and in the pelvis. Histologically, there was direct transition from benign epithelium in the ectopic endometrial glands, to dysplastic and neoplastic tissue. In different areas, the tumor included elements of yolk sac type (endodermal sinus tumor), choriocarcinoma, and teratoma. The implications of this unique transformation and combination are discussed briefly.

Assessment of proximity of a wound to major vascular structures as an indication for arteriography.

The use of angiography to evaluate penetrating extremity wounds with proximity to major vascular structures remains controversial. Arteriography in the asymptomatic patient with a penetrating extremity wound is reported to identify arterial injuries in 6% to 21% of patients; however, some injuries may have little clinical importance. This study attempted to determine the value of proximity as an indication for angiography. Five hundred seven asymptomatic patients with 534 penetrating extremity injuries underwent arteriography due to proximity to major vascular structures. Thirty-six arteriograms (6.7%) were positive. Seven patients did not undergo operative exploration, 19 patients (3.6%) had arteriograms, and ten (1.9%) had false-positive arteriograms. The remaining 498 patients had true-negative examination results. Arteriography was associated with 13 complications (2.6%). Proved vascular injury in the clinically asymptomatic patients in our series was extremely low (3.6%). These data make it difficult to justify arteriography due to proximity of injury to major vascular structures. However, it is difficult to abandon exclusion arteriography based on these retrospective data. These observations do suggest that better criteria to define proximity need to be identified.

Myelin basic protein interacts with the myelin-specific ganglioside GM4.

Demyelinated plaques in multiple sclerosis have decreased amounts of both myelin basic protein and sialosylgalactosylceramide (GM4), a ganglioside specifically localized in myelin and oligodendroglia of the central nervous system. We have found that myelin basic protein is capable of releasing large quantities of entrapped [14C]glucose from multilamellar liposomes containing GM4. If the conformation of GM4 in liposomal membranes resembles that of GM4 in its natural environment, basic protein and GM4 may be associated within the myelin sheath of the central nervous system and their interaction altered in demyelinating diseases such as multiple sclerosis.

Sarcoidosis in coroner's autopsies: a critical evaluation of diagnosis and prevalence from Cuyahoga County, Ohio.

BACKGROUND AND AIM OF THE WORK: During a histologic study of sarcoidosis in tissues made available from the Cuyahoga County Coroner's autopsy files, the prevalence of this disorder seemed much greater than generally reported. Aims were to evaluate this impression and if confirmed, to explore underlying reasons, including reliability of diagnoses and possible overestimates as well as possible associations with particular population subgroups. METHODS: Cases were retrieved by computer search of diagnoses coded over the past 7 years. Extent of organ involvement was documented and relevant microscope slides reviewed, with consideration of diagnostic criteria generally proposed. Clinical information, occupational risk and evidence for illicit drug use were evaluated. Acceptable cases were categorized by gender, race and age. Crude and standardized prevalences were calculated and related to 1990 US Census figures for the County. RESULTS: On review of 9324 adult autopsies performed for medico-legal reasons, 31 were judged to have sarcoidosis. Standardized prevalence was 320 +/- 72/10(5). Among population subgroups statistically significant differences were not established. However, crude prevalences suggested that females, blacks and younger persons were more likely to have sarcoidosis than males, whites and older subjects, with ratios of 1.6 to 1; 4.7 to 1; and 2.5 to 1, respectively. Overall prevalence was some 10 times greater than indicated from State-wide death certificates. CONCLUSIONS: Sarcoidosis as found in cases autopsied at the/Coroner's Office, Cuyahoga County, is much more prevalent than generally reported. It is an under-appreciated cause of sudden unexpected death. Young black females appear to be most affected, and worthy of further investigation.

Sizing of crimped Dacron grafts.

The aim of this study was to correlate the stated size of Dacron grafts (Microvel) with their actual internal diameter and to compare this with measurements by ultrasound in the early postoperative period. Grafts of stated diameters of 7, 8, 9, and 10 mm were studied. Grafts were measured by graded probes and by measuring the width of the longitudinally opened graft and calculating the diameter. Each graft accepted a probe 1 mm larger than its stated size very easily and 2 mm greater when stretched. By open measurement, the grafts were 1.3 to 1.8 mm greater than their stated diameter without stretching. Twenty grafts were studied by duplex ultrasound for diameter and peak systolic velocity within 3 months of implantation. The grafts were 12% larger than their stated graft size. Some of the larger grafts showed low velocity and wall thrombus. We conclude that Microvel grafts are larger than their stated diameter. The same size discrepancy was seen in in vitro measurements of Vascutek (Dacron) grafts but not in polytetrafluoroethylene (Gortex) grafts. Since flow velocity is related to the diameter of the graft, this information should be useful when choosing the diameter of a prosthetic vascular graft.

Prosthetic-vein composite graft with remote popliteal arteriovenous fistula.

BACKGROUND: Inadequate autogenous vein is often the limiting factor in femoral-distal reconstruction. Sequential grafts, distal arteriovenous fistulae (AVF), and autogenous cuffs have been recommended in this setting. This paper describes a new method of composite reconstruction, enhanced with a popliteal AVF. PATIENTS AND METHODS: Data was collected prospectively on 13 patients (14 limbs) who presented with severe ischemia (rest pain or ulcer 6, gangrene 8). The average age was 75 years. Eleven of the reconstructions were to tibial outflow tracts. The novelty of the technique is in the construction of a controlled AVF between the upper end of an autogenous vein graft and the popliteal vein. An uncomplicated autogenous anastomosis is created distally. The synthetic graft is placed between the common femoral artery and the vein graft distal to the AVF. The flow is balanced between the fistula and the outflow tract by narrowing the fistula while monitoring the pressure in the graft system. RESULTS: Patency was assessed by color duplex scan. Two grafts occluded (at 5 and 7 months) and 1 was revised at 12 months. Mean peak systolic velocity in the synthetic portion was 130 cm/s. Limb edema has not been a problem. There were 2 perioperative cardiac deaths. Mean follow up is 15 months (range 6 to 36). CONCLUSION: The addition of a controlled AVF below the synthetic portion of a composite graft appears promising when vein is in limited supply.

Pelvic recurrence after anterior resection and EEA stapling anastomosis for potentially curable carcinoma of the rectum.

Rectal carcinoma can be treated by anterior resection with EEA anastomosis in order to preserve rectal continuity in those patients in whom anastomosis may be technically difficult. In our initial local experience however, the pelvic recurrence rate has been approximately three times as high as would be expected. The surgical results were good in those patients with Dukes' B lesions. The majority of failures occurred in those with Dukes' C lesions. When the preoperative assessment indicates the strong likelihood of a Dukes' C lesion, consideration of abdominoperineal resection must be given if cure is anticipated. Most surgeons will obtain a more complete pararectal tissue clearance with this procedure than with anterior resection. Those who wish to preserve rectal continuity with curative procedures for rectal carcinoma must become proficient at pararectal tissue clearance if a low rate of pelvic recurrence is to be achieved. The EEA stapler can enable safe anastomosis when these other factors have been accomplished. When cure is anticipated, it can only be provided for the majority of patients at initial surgical resection. The EEA stapler has a unique value in patients who have resectable rectal carcinoma, and yet have distal metastasis by the time initial surgery is performed. The preservation of rectal continuity in such patients is an excellent method of palliation.

An aggressive local approach to vascular graft infection.

PURPOSE: To present the use of sartorius myoplasty (SM) and superficial femoral vein (SFV) in a graft-sparing approach to vascular graft infection. METHODS: Twenty-five patients were treated for Szilagyi grade III groin infections during the last 10 years. Fifteen presented early (E), mean 2 months; 10 late (L), mean 6.5 years. Bacteria E/L: Staphylococcus epidermidis 1/7, S aureus 6/0, other gram positive 1/0, gram negative 4/1, mixed 6/0 (one pseudomonas). There were 13 aortofemoral (AF), 5 crossover, and 7 femoral distal reconstructions. SM was used to cover exposed grafts after radical debridement. When the graft was free-floating or bleeding, this segment was removed and replaced with SFV. The remaining infection was controlled with antibiotics. RESULTS: One of 10 patients treated by SM alone required SFV replacement for bleeding. Four of 9 AFs treated by partial SFV replacement +/- SM had persistent infection treated by complete graft removal in 3. Six grafts were removed electively in lower risk situations. There was no perioperative mortality, and no early or late limb loss. CONCLUSIONS: This experience supports an attempt at aggressive local treatment in frail patients.

Color flow duplex screening of infrainguinal grafts combining low- and high-velocity criteria.

The aim of this prospective study was to evaluate the ability of duplex ultrasonography to identify infrainguinal grafts at high risk for failure. The criteria used identified low flow by low peak systolic velocity (less than 45 cm/s) and stenosis by high velocity (greater than 300 cm/s) or by velocity at the stenosis three times the velocity in the adjacent normal graft. A total of 114 patent grafts were scanned and compared with concurrent angiograms. Duplex scanning correctly identified 18 high-risk grafts by low-flow criteria and an additional 21 by stenosis criteria. There was one false-negative finding (sensitivity 98 percent). The velocity ratio of the stenosis to the adjacent graft was useful in estimating the degree of stenosis. Color flow duplex imaging reduced examination time through visual feedback by highlighting the graft and areas of high velocity. These results indicate that color flow duplex scanning, combining low- and high-peak systolic criteria, is a very sensitive screening test in the early detection of failing grafts.

The Brenner tumor: a report of multiple tumors.

A case of multiple Brenner tumors was studied with light and electron microscopy. Among the epithelial elements, argyrophil cells were found in addition to the typical "transitional" and mucus-secreting cells. In some areas, it was not possible to distinguish epithelial from tumor stromal cells. By electron microscopy, dense-core secretory granules were identified, as well as previously undescribed cytoplasmic tubular bodies. Some epithelial cells were intimately associated with reticulin and collagen. Some stromal cells had desmosomes, dense bodies, incomplete basal lamina, and, occasionally, multivesicular bodies, which suggests epithelial differentiation. Previous authors have documented morphologic similarities and continuity between Brenner-tumor epithelium and ovarian cells of almost every type. The inference is that there are multiple possible sources for the Brenner tumor. Classification must therefore be based on realize differentiation rather than presumed cell of origin.

A measurement model of cortisol reactivity of healthy older adults.

Research on the physiological adaptation process has found that stress is associated with the rate of cortisol secretion, the main hormone that reflects stress. However, considerable variation among subjects has been reported. Using a sample of older adults (N=46), we tested the hypothesis that cortisol reactivity is composed of (1) a situation-related component representing hypothalamic influence on cortisol secretion observed on three different occasions, and (2) a stable component representing a general trait responsible for cortisol responses observed from occasion to occasion. LISREL VIII was used to test this hypothesis. Results indicated that a homogeneous reliability model was not supported by the data. A congeneric measurement model represented a better fit to the data. Results suggest that subjects have consistent patterns of response during separate experimental occasions. However, results do not suggest a consistent pattern of response over time. The main implication of these results is that salivary cortisol measures are sensitive to experimental stress situations. As such, this noninvasive method may be useful in examining adaptive responses to stress.

Harvest of brachio-basilic vein complex for arterial bypass.

Brachial vein has been overlooked as a conduit for arterial bypass. It can be harvested with very little morbidity. A deep incision placed on the medial aspect of the upper arm allows exposure of the brachial and basilic veins, giving access to vein of adequate length and diameter for many applications. The medical deep veins of the upper arm are usually our first alternative to ipsilateral saphenous vein for autogenous reconstruction. Because of inconsistent anatomy and nomenclature in this area, we refer to these veins as the "deep brachial vein complex".