Harvard Medical School

Protocollagen proline hydroxylase was purified from chick embryos, and it was examined by electron microscopy with negative staining techniques. Enzyme prepared in Helsinki consisted almost entirely of single ring structures. Enzyme prepared in Philadelphia under essentially the same conditions consisted almost entirely of tubular forms in which four rings were stacked together. Repeated freezing and thawing of the Helsinki preparation markedly increased the number of four-ring structures, but it was not possible to establish reproducible conditions for the interconversion of these two major forms of the enzyme.

Each ring in the four-ring structure had a diameter of 100 A, a height of 30 to 35 A, and a central hole of about 20 A. The single ring structure was slightly more distended. The molecular weight of each ring was about 200,000, and therefore the molecular weight of the four-ring structure was about 800,000.

The inner two rings of the four-ring structure were closer together than the outer two rings. Since the four rings appeared to be identical, the observations suggested that the rings were held together by a combination of heterologous and isologous bonds. No regular structures with more than four rings were seen, and the simplest bonding sequence consistent with these observations was (AB)—(AB)— (BA)—(BA) in which larger structures were not favored, because the formation of the inner B—B bond altered the A surfaces on the inner rings so that they were not equivalent to the A surfaces on the outer rings.

Previous reports demonstrated that synthetic polytripeptides of the structure (Pro-Pro-Gly)10, prepared with a specific modification of the Merrifield technique, formed triple helical structures in aqueous solution which were similar to the triple helical structures of tropocollagen. The studies presented here were undertaken in order to answer the question of whether the individual polytripeptide chains within the triple helices were packed in a parallel fashion similar to vertebrate collagen or in an antiparallel fashion similar to the structure which has been suggested for collagen from the cuticle of the round worm, Ascaris lumbricoides. It was found that electrostatic interactions between the COOH-terminal and NH-terminal groups of (Pro-Pro-Gly)10 could be used to probe the detailed structure of triple helices formed by the polytripeptide.

Titration of (Pro-Pro-Gly)10 in triple helical conformation indicated that the pK of the NH-terminal group was 0.59 units lower than the pK for the same group in nonhelical (Pro-Pro-Gly)5. The titration of the NH-terminal group of helical (Pro-Pro-Gly)10 was significantly broader than the theoretically predicted curve, and the pK increased after the polytripeptide was heated and quenched.

Furthermore, the melting curve for the helix coil transition of (Pro-Pro-Gly)10 was changed by protonation of the NH-terminal or COOH-terminal groups. The Tm for the dipolar ion form was about 10° lower than for either the fully protonated or unprotonated forms of the helical polytripeptide.

The results indicate that in the triple helical structure formed by (Pro-Pro-Gly)10 the three chains are in register and parallel.

"Janusian thinking"—the capacity to conceive and utilize two or more opposite or contradictory ideas, concepts, or images simultaneously—is discussed in relation to its role in the creative process in art, literature, architecture, music, science, and mathematics. I feel that understanding the psychological factors in creativity should be of importance in the theory and everyday practice of the art of psychotherapy.

Created five years ago, the Regional Medical Programs now have established organizations that cover the entire country. Private medicine is involved to a degree unparalleled in any other federal health program. In many cases the Programs have served to bring together elements of the health-care delivery system that have previously been isolated from one another. In other cases they have promoted the extension of recent medical advances out from the medical centers, improving the care given by private practitioners and community hospitals. Yet confusion exists over the mission of the Regional Medical Programs, and the means by which to achieve that mission. Particularly at issue is their role in changing, rather than simply upgrading, the existing health-care delivery system. It remains to be seen whether they are faced with a golden opportunity or an insoluble dilemma.

A noninvasive technique for evaluating the extent of myocardial ischemic injury after experimental coronary artery occlusion was devised and applied to study alterations in the extent of injury produced by hemodynamic and pharmacologic interventions. The technique was then extended to the assessment of myocardial ischemic injury in patients with acute myocardial infarction. In 7 closed chest dogs, electrocardiograms were recorded from 15 sites on the chest wall before and after intermittent occlusions of the left anterior descending coronary artery. There was no S-T segment elevation before the occlusion; 15 minutes after occlusion the sum of S-T segment elevations (ΣS-T) averaged 15.0 ± 3.0 mm (SEM, 1 mm deflection = 0.1 mv), and an average of 4.2 ± 0.6 sites exhibited elevations exceeding 0.1 mv (NS-T). Occlusions occurring during administration of isoproterenol (0.25 μg/kg per min) increased ΣS-T to 51.0 ± 9.0 mm and NS-T to 10.6 ± 0.9, whereas occlusions occurring after administration of propranolol (1 mg/kg) decreased ΣS-T to 3.0 ± 1.5 mm and NS-T to 0.2 ± 0.2. In 8 dogs the extent of ischemic injury, manifested by S-T segment changes, was decreased by propranolol and norepinephrine and increased by hemorrhagic hypotension and isoproterenol, applied up to 6 hours after occlusion. Reproducible S-T segment maps, using 35 surface electrodes, were obtained in 19 patients with acute myocardial infarction. In 15 patients studied serially, ΣS-T decreased from 54.25 ± 7.00 to 38.50 ± 6.30 mm and NS-T from 18.7 ± 2.5 to 12.3 ± 2.8, respectively, during a 24 hour period. However, in 3 patients in whom ventricular fibrillation, arterial hypotension and further ischemic pain occurred, ΣS-T and NS-T increased whereas in another patient propranolol decreased ΣS-T and NS-T. Thus, precordial mapping, both in dogs and patients, shows changes parallel to those measured by the epicardial technique and should provide a useful clinical tool for determining acute changes in the extent of ischemic injury.

The effects of coronary artery reperfusion 3 hr after coronary occlusion on contractile function and the development of myocardial damage at 24 hr was studied experimentally. In 14 control and 6 reperfused dogs, relationships between epicardial ST segment elevation 15 min after coronary occlusion and myocardial creatine phosphokinase activity (CPK) and histologic appearance 24 hr later were examined. The electrocardiograms were recorded from 10 to 15 sites on the left ventricular epicardium and transmural samples for CPK and histology were obtained from the same sites where epicardial electrocardiograms had been recorded. An inverse relation existed between ST segment elevation (mv) 15 min after occlusion and log CPK activity (IU/ mg of protein) 24 hr later, log CPK = - 0.06ST + 1.26. In dogs subjected to coronary artery reperfusion, there was significantly less CPK depression (log CPK = - 0.01ST + 1.31, [P < 0.01]) than that expected from the control group. In the control group 97% of specimens showing ST segment elevations over 2 mv at 15 min showed abnormal histology 24 hr later. In contrast, in the reperfused group 43% of sites exhibiting elevated ST segment at 15 min showed abnormal histology 24 hr later. In six additional dogs it was shown that the paradoxical movement of the left ventricular wall could be reversed within 1 hr of perfusion. Therefore, by enzymatic and histologic criteria, as well as by functional assessment, coronary artery reperfusion 3 hr after occlusion resulted in salvage of myocardial tissue.

The localization of 3H-labeled cholesterol in nerves undergoing degeneration and regeneration was studied by radioautography at the electron microscope level. Two types of experiments were carried out: (a) Cholesterol-1,2-3H was injected intraperitoneally into suckling mice. 5 wk later, Wallerian degeneration was induced in the middle branch of the sciatic nerve, carefully preserving the collateral branches. The animals were then sacrificed at various times after the operation. During degeneration, radioactivity was found over myelin debris and fat droplets. In early stages of regeneration, radioactivity was found in myelin debris and regenerating myelin sheaths. Afterwards, radioactivity was found predominantly over the regenerated myelin sheaths. Radioactivity was also associated with the myelin sheaths of the unaltered fibers, (b) Wallerian degeneration was induced in the middle branch of the sciatic nerves of an adult mouse, preserving the collateral branches. Cholesterol-1,2-3H was injected 24 and 48 hr after the operation and the animal was sacrificed 6 wk later. Radioactivity was found in the myelin sheaths of the regenerated and unaltered fibers. The results from these experiments indicate that: (a) exogenous cholesterol incorporated into peripheral nerve during myelination remains within the nerve when it undergoes degeneration. Such cholesterol is kept in the myelin debris as an exchangeable pool from which it is reutilized for the formation of the newly regenerating fibers, especially myelin. (b) exogenous cholesterol incorporated into the nerves at the time that degeneration is beginning is also used in the formation of new myelin sheaths during regeneration, (c) mature myelin maintains its ability to incorporate cholesterol.

Since isoproterenol, tachycardia induced by atrial pacing, propranolol and Hyaluronidase alter the extent and severity of cardiac damage after coronary occlusion, their effects on the function of the severely ischemic heart were studied. In 40 dogs, blood from the femoral artery perfused the cannulated left coronary artery with flow controlled by a roller pump. Total left coronary blood flow was reduced progressively every 3 minutes from a control value of 84.2 ml/min per 100 g of left ventricular myocardium until mean left atrial pressure exceeded 20 mm Hg. In control runs, coronary blood flow decreased by 49 percent; with infusion of isoproterenol (0.08 μg/kg per min) coronary blood flow was reduced by only 24 percent (no. = 12, P < 0.01); after pretreatment with propranolol (1 mg/kg) coronary blood flow could be reduced by 55 percent (no. = 8, P < 0.05); and after pretreatment with Hyaluronidase (2,000 National Formulary units/kg) coronary blood flow could be reduced by 65 percent (no. = 6, P < 0.01) until left atrial pressure increased to 20 mm Hg. With coronary blood flow at 67 ml/min per 100 g, that is, at 75 percent of control value, left atrial pressure averaged 4.9 mm Hg at a heart rate of 120 beats/min and increased to 12.7 mm Hg (no. = 9, P < 0.01) at a heart rate of 165 beats/min. Thus, contrary to the effects of isoproterenol and tachycardia in the normal and failing nonischemic heart, these interventions depress left ventricular function in the severely ischemic heart, whereas propranolol and Hyaluronidase improve this function. The effects of these interventions on ventricular function parallel those produced by these same interventions on the extent and severity of myocardial injury in the presence of coronary occlusion.

The influence of the administration of pharmacologic doses of hydrocortisone on the extent and severity of acute myocardial ischemic injury and on subsequent necrosis after acute coronary occlusion was investigated in 28 dogs. In order to study acute myocardial injury, repeated epicardial electrocardiograms were recorded from 10 to 15 sites on the anterior surface of the left ventricle. Average ST segment elevation (ST) and the number of sites in which ST segment elevation exceeded 2 mV (NST), indices of the magnitude and extent of myocardial injury, respectively, were analyzed at 30 and 60 min after coronary occlusion. In the control group ST and NST did not change significantly in this time interval while in the treated group, which received 50 mg/kg hydrocortisone just after the 30 min recording, ST fell from 3.5+/-0.8 to 1.1+/-0.4 mV (P 2 mV) in the control group showed histologic changes compatible with early myocardial infarction in 96% of specimens, while this occurred only in 61% and 63% of specimens, respectively, in the treated groups, showing that over one third of the sites were protected from undergoing necrosis due to the intervening hydrocortisone treatment. Thus pharmacological doses of hydrocortisone prevent myocardial cells from progressing to ischemic necrosis even when administration is initiated 6 h after coronary occlusion.

Physician assistants used problem-oriented protocols in management of patients with diabetes and hypertension. The protocols directed the physician assistant in determining what data to collect, and whether the patients required a physician's care at that visit. Physicians verified the ability of the physician assistants to collect history and physical-examination data accurately, noting false-positive findings on 11 per cent of 381 visits, and false-negative findings on 1 per cent of visits. Subsequently, physicians selected certain patients as "suitable" to be managed by the physician assistants. "Suitable" patients were then randomly assigned to be seen by the physician assistants (experimental visits) or physicians (control visits) on return visits. On 37 percent of experimental visits over an 11-month period, the patients went home without seeing a physician. Physicians accepted the protocol disposition decision in 94 per cent of the cases. Patients accepted the system, as reflected by their attendance rate at the clinic.

Glutaraldehyde-carbohydrazide polymer (GACH) was used to embed olfactory tracts, trapezoid body, and sciatic nerves of 9-, 10-, and 49- day old mice 2 h, 24 h, and 6 wk (respectively) after the intraperitoneal administration of [1,2-3H]cholesterol. Greater than 94% of radioactive cholesterol was retained in the GACH-infiltrated brain 24 h or more after injection. The fine structural preservation of both central and peripheral nervous tissues was excellent. Quantitative analysis of electron microscope autoradiographs demonstrated that [1,2- 3H]cholesterol is limited to blood vessel walls and lumen within the central nervous system at 2 h and 6 wk postinjection, but neurons and neuropil also contain the labeled cholesterol. The thickest myelin sheaths in the adult mice appear to be uniformly labeled throughout their width. No relationship of the retained [1,2-3H]cholesterol to the node of Ranvier was found in the adult sciatic nerve.

Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin- labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.

Two improved procedures were developed for activating ferritin so that the ferritin could be covalently linked to antibodies. One procedure involved use of a water-soluble carbodiimide and N-hydroxysuccinimide to prepare ferritin-containing activated esters. The other involved activation of the ferritin with excess glutaraldehyde. The ferritin- antibody conjugates prepared with the two procedures were shown to have a number of properties which made them suitable for locating antigenic components in cells.

Three distinct antiprocollagen preparations were characterized and used in immunocytochemical staining of chick embryo corneal and tendon cells. The several ferritin-conjugated antibody preparations permitted similar location of procollagen in the cisternae of the rough endoplasmic reticulum and in Golgi elements in both cell types. The ability to demonstrate and interpret specific ferritin staining was dependent on the extent of membrane breakage in each of those organelles, coupled with adequate retention of cell morphology. Corneal fibroblasts appeared to suffer more extensive intracellular membrane damage under controlled conditions of homogenization than tendon fibroblasts, facilitating the identification of procollagen in Golgi vacuoles of these cells. None of the labeled material appeared to by cytoplasmic in origin since ferritin was observed in the cytoplasm only in the vicinity of Golgi elements that were extensively broken. This study extends previous immunological evidence for the presence of procollagen in the Golgi complex and calls attention to the problems to be encountered in locating the antigen in small Golgi vesicles and lamellae.

To the Editor.— The recent warning in The Medical Letter1 regarding the mutagenicity and possible carcinogenicity of metronidazole (Flagyl) raises the question of whether there is an alternative treatment of equal efficacy that is not equally suspect.The pharmacologic treatment options seem to include orally administered nitroimidazole drugs other than metronidazole, or topical agents available in vaginal-tablet or suppository form, such as furazolidone and nifuroxime (components of Tricofuron). None of the other nitroimidazoles are currently available in the United States, although tinidazole (Fasigyn) will apparently soon be released. Tinidazole2 and nimorazole are reported to have a better than 90% cure rate. However, the mutagenicity of nimorazole was demonstrated in the same study that implicated metronidazole,3 and subsequent research by the same group has apparently also demonstrated the mutagenic potential of tinidazole (C. E. Voogd, unpublished data).The efficacy of topically applied furazolidone, alone or in combination with