[Use of the Nd:YAG laser in otorhinolaryngology].

The Nd:YAG laser is suitable for the treatment of various otorhinolaryngological clinical disorders. These include the palliative reduction of tumor size in sites with difficult access, treatment of hemangiomas and reduction of hyperplastic turbinates. Within certain limits, other indications are treatment of recurrent epistaxis and recurrent polyposis. Palliative reduction of malignancies in the nasopharynx, esophagus, and bronchial system (laser power density: 1500-8000 W/cm2) must sometimes be carried out in several sessions in order to avoid complications due to the laser (i.e., perforation of the bronchial or esophageal wall, lesions of adjacent vessels or nerves). Nd:YAG laser treatment of hemangiomas (500-3000 W/cm2) can lead to excellent results. To avoid excessive thermal lesions, vascular tissue is cooled with ice cubes or with an ice-cold Ringer's solution. The laser process is continued until the onset of tissue blanching. Carbonizations of the tissue are to be avoided. In Nd:YAG laser therapy of hyperplastic lower nasal conchae (approx. 1000 W/cm2), results are based on submucous scarring in which the covering epithelium is maintained. The objective of Nd:YAG laser treatment of recurrent epistaxis in patients with Osler's disease (500 W/cm2) is to reduce the incidence of hemorrhage. Use of the laser in recurrent polyposis is best confined to patients who refuse conventional surgical revision operations. Laser light (500-3500 W/cm2) should only be applied for a short period of time (0.5 s) to avoid creating a rarefying osteitis.


[Sonography and plain roentgen image in diagnosis of salivary calculi--experimental studies].

A model was developed in which high-resolution real-time sonography was compared with radiological examinations for determining their sensitivity in diagnosing salivary gland stones. For this purpose an artificial salivary gland medium was compared with human salivary gland tissue for echogenicity. It was shown that both the artificial and the true salivary gland medium possessed comparable acoustic properties. Forty salivary gland stones were examined ultrasonographically in both media with 5 and 7.5 MHz linear scanners and a 5 MHz sector scanner, following which a plain X-ray was done, while 35 of 40 calculi could be definitely identified with linear scanners in both media, this could be accomplished by X-ray only in 50% (20 of 40 calculi). All salivary gland stones greater than 1-2 mm could be detected ultrasonographically, whereas radiological identification did not correlate with stone size. Mineral composition was found to influence the radiological study but not sonographic pictures of sialoliths. If sialolithiasis is suspect clinically, ultrasonography with a linear scanner must be considered the diagnostic procedure of choice.


[Esophagotracheal fistula of the cervico-thoracic area--problem solution using a combined trans-cervical and transthoracic approach].

Closure of oesophagotracheal fistulae in the cervicothoracic region using a cervical approach often fails. We report a patient with carcinoma of the larynx who underwent pharyngolaryngectomy, postoperative radiotherapy and several operations for reconstruction of the hypopharynx and cervical oesophagus elsewhere. Finally we were asked to close an oesophagotracheal fistula in the cervicothoracic region. We were successful using a pectoralis major muscle flap via a combined transcervical-transthoracal approach. This interdisciplinary procedure seems to be the method of choice in difficult cases of achieving safe closure of fistulae in this region.


[Histocochleographic studies of the late toxicity of cisplatin].

The aim of the investigation was to determine in an animal model the time required for cochlear changes to appear after cisplatin therapy, i.e. whether there is any late ototoxicity detectable. For this purpose we performed histochochleographic studies in the laboratory rat. In two trials different groups of 6-7 rats were given a single daily dose of 2 mg/kg body wt. cisplatin over 5 days. After various periods of time surface preparations of the organ of Corti were performed. The results obtained showed that cochlear damage was evident as early as 4 days after the final injection of drug and no further significant changes in cochlear damage could be seen during the period of observation.


[Leiomyosarcoma of the mouth floor and oropharynx].

Leiomyosarcomas arise rarely in the head and neck. We report a 41-year-old woman with an extremely rare leiomyosarcoma of the floor of the mouth extending to the oropharynx. This article stresses the value of imaging methods such as CT, MRI and ultrasound for preoperative diagnosis, and emphasises histopathological findings. The therapeutic principles and prognostic factors of leiomyosarcomas are illustrated on the basis of this case.


Clinical findings in cats with dilated cardiomyopathy and relationship of findings to taurine deficiency.

Between October 1986 and September 1988, 37 cats with moderate to severe idiopathic myocardial failure (dilated cardiomyopathy) were evaluated prospectively. Low plasma taurine concentration and diet history including foods that can cause taurine deficiency were documented in most of the cats. Comparison with a retrospectively studied population of 33 cats with dilated cardiomyopathy diagnosed between 1980 and 1986 demonstrated that the clinical and historical findings in the 33 retrospectively studied cats were similar to those in the 37 cats studied prospectively. Clinical findings in the 2 groups were also similar to findings previously reported in the literature. Because clinical findings and diet history were similar in the prospective and retrospective groups, we believe that many cats in the latter group had diet-induced taurine deficiency. These findings support the conclusion that most cases of dilated cardiomyopathy in cats have a common etiopathogenesis related to diet and as such are preventable.


Response of cats with dilated cardiomyopathy to taurine supplementation.

Between October 1986 and September 1988, 37 cats with moderate to severe idiopathic myocardial failure (dilated cardiomyopathy) were evaluated. Clinical management of these cats was similar to that described in the literature, except that it also included administration of 500 or 1,000 mg of the sulfur amino acid, taurine per day. Early death (death within the first 30 days of treatment) occurred in 14 (38%) cats. One cat was lost to follow-up evaluation. Twenty-two cats (59%) had marked clinical and echocardiographic improvement and survived longer than 240 days. In all but 1 cat, the observed improvement in echocardiographic measurements persisted. Hypothermia and thromboembolism were positively associated with an increased risk of early death. Administration of digoxin did not significantly affect survival. All 22 cats that survived greater than 30 days remained clinically stable despite withdrawal of all medications except taurine. Administration of taurine was eventually discontinued in 20 of the 22 cats and adequate taurine intake was thereafter provided for in the food. The clinical response and 1-year survival rate of 58% (21 of 36 cats with a known outcome) in the taurine-treated group represents a marked improvement, compared with a 1-year survival rate of 13% (4 of 31 cats with a known outcome) in a retrospectively evaluated population of 33 cats with dilated cardiomyopathy.


Renal transplantation for treatment of end-stage renal failure in cats.

Renal transplantation was performed as treatment of end-stage renal failure in 23 cats. Twenty-two cats had chronic renal disease and 1 cat had acute renal disease associated with ethylene glycol-induced toxicosis. Sixteen cats were discharged from the hospital. Nine survived a mean of 8.4 +/- 6.5 months, and 7 cats continue to survive at the time of this report (mean 12.6 months). Seven cats died within 2 weeks of surgery. All renal allografts were obtained from unrelated blood-crossmatch-compatible donors. No deaths were attributable to acute renal allograft rejection, demonstrating the successful maintenance of renal allografts by use of cyclosporine and prednisolone immunosuppression in cats.


Selenium toxicosis in feeder pigs.

Selenium toxicosis was diagnosed in feeder pigs on a central Michigan farm. Use of a commercial supplement, found to contain approximately 20 times the intended Se concentration, resulted in a Se concentration of 8.1 mg/kg of the complete feed. This was fed for 34 days during which daily feed consumption decreased approximately 35%, several pigs developed weakness and forelimb paresis, and 1 pig died. The highest serum Se concentration measured was 1,550 ng/ml (normal range, 140 to 190 ng/ml). Normal feed consumption returned when an alternative feed was provided. Mean serum Se concentrations of representative pigs, monitored over the subsequent 26 days, decreased from 905 to 258 ng/ml. Histologic examination of a recovering pig revealed skeletal and cardiac myopathy and bilaterally symmetric malacia of the gray matter of the ventral horns of the spinal cord. During the developing toxicosis, the pigs consumed an estimated 11.4 mg of Se/pig/d.


Dioxin intoxication from chronic exposure of horses to pentachlorophenol-contaminated wood shavings.

Investigations into the cause of health problems on a horse-breeding farm led to the discovery of high concentrations (630 to 9,810 mg/kg of bedding) of pentachlorophenol in wood shavings used as bedding for horses over a period of 2 to 4 years. Toxicologic signs in the horses were characteristic of toxic effects associated with exposure of polychlorinated dibenzo-p-dioxins and dibenzofurans. Tissue residue analysis confirmed presence of toxic polychlorinated dibenzo-p-dioxin and dibenzofuran isomers known to be in pentachlorophenol, substantiating the bioavailability of polychlorinated dibenzo-p-dioxins and dibenzofurans in the wood shavings. The findings provide evidence that residue concentrations in the range of 2 ng/g of toxic polychlorinated dibenzo-p-dioxin isomers in liver or fat correlate with toxicologic effects in horses.


Heterotopic salivary tissue in a weanling colt.

Heterotopic salivary tissue was diagnosed in a 7-month-old Quarter Horse colt with a history of a draining wound in the left temporal region from time of birth. Surgical excision of the tract was performed for diagnosis and treatment. Histologically, tissues were compatible with a mixed-type salivary gland, most likely of parotid salivary gland origin. Complete resolution of the drainage was achieved after surgical removal of the ectopic tissue.


Ultrasonographic evaluation and surgical treatment of humeral osteitis and bicipital tenosynovitis in a horse.

Ultrasonography and radiography were used to diagnose bicipital tenosynovitis and humeral osteitis in a horse with a history of lameness of 7 weeks' duration. Surgical exploration confirmed the diagnosis.


Immunodeficiency associated with lymphosarcoma in a horse.

Immune system dysfunction and immunoglobulin deficiency was diagnosed in a 2-year-old horse with disseminated lymphosarcoma. Prolonged (35 days) parenteral nutrition was delivered to support the horse during a period in which immune function studies could be performed. Correction of nutritional compromise by use of parenteral nutrition did not correct the immunoglobulin deficiency, and results of lymphocyte phenotype testing did not indicate abnormal proportions of leukocytes. Lymphoblast transformation studies were suggestive of a circulating immunosuppressive factor in the horse's serum. Normal cell function was detected when the cells were stimulated in precolostral equine serum.


Multicentric cutaneous pythiosis in a foal.

A 5-month-old male Quarter Horse was examined because of multicentric, granulomatous, cutaneous lesions of the dorsum and prepuce. The cutaneous lesions were excised, and sodium iodide was administered orally for 2 weeks. Multicentric cutaneous pythiosis was diagnosed by histologic examination and immunoperoxidase staining of excised tissues. By 3 months after surgery, all lesions had healed.


Idiopathic granulomatous disease with ocular adnexal and cutaneous involvement in a dog.

Idiopathic granulomatous disease was the cause of bilateral eyelid masses and additional ocular adnexal disease in a dog. Histologically similar granulomas developed concurrently in distant sites including the limbs, prepuce, and testicles. Periodic resolution and redevelopment of the masses were observed and were unaffected by corticosteroid treatments. Rapid diminution of some of the masses was found after treatment with polyethylene glycol modified L-asparaginase, but poor client compliance and episodes of spontaneous resolution of some of the masses made it difficult to accurately assess effectiveness of treatment. Clinicians should be aware that ocular adnexal granulomas may simulate neoplasms and be a component of a more widespread disease process.


Severe bilateral fibrosing pleuritis associated with chronic chylothorax in five cats and two dogs.

Severe bilateral fibrosing pleuritis was diagnosed in 5 cats and 2 dogs with chronic chylothorax. All animals were dyspneic on initial examination and remained moderately to severely dyspneic after thoracentesis. Radiographic evidence of fibrosing pleuritis included rounded lung lobes and failure of the lungs to reexpand following effective pleural drainage. Fibrosing pleuritis was also suggested in several animals with radiographic evidence of pleural fluid, in which pleural fluid could not be retrieved. Macroscopically, the lung lobes of all animals were compressed and atelectatic to various degrees, and the pleura appeared to be diffusely thickened and roughened. In several animals, fibrous adhesions were found between the parietal and visceral pleura of some lobes. Lung lobes were markedly constricted and appeared as small, smooth, rounded hilar masses in 4 animals. Mild to moderate pulmonary edema was evident in 3 animals at necropsy. Six of the 7 animals died (2) or were euthanatized (4) within 72 hours after the diagnosis of fibrosing pleuritis. The fibrosing pleuritis developed in 1 animal with lymphoblastic lymphosarcoma and chylothorax after treatment via passive pleuroperitoneal drainage; this animal was euthanatized because of underlying neoplasia. One cat, in which decortication was performed and resulted in marked reexpansion of the lung lobes, died 4 hours after surgery with signs compatible with pulmonary edema. On the basis of our findings, we suggest that animals with chronic chylothorax are at risk to develop fibrosing pleuritis. Furthermore, animals with severe bilateral fibrosing pleuritis should be given extremely guarded prognoses.


Paraphimosis in a teaser bull with penile translocation.

A bull with penile translocation and bilateral caudal epididymectomy developed paraphimosis when used for estrus detection of cows. The paraphimosis did not resolve with bandaging and topical treatment; thus, it was surgically corrected by retracting the distal sigmoid flexure of the penis and suturing it to the superficial fascia in the medial femoral region. Paraphimosis should be considered a possible sequela to penile translocation in bulls.


Chronic frontal sinusitis in dairy cattle: 12 cases (1978-1989).

Chronic frontal sinusitis in 12 dairy cattle most often was associated with a history of dehorning, in which the sinus was entered (67%), or with respiratory tract disease (25%). The most common organisms isolated were Actinomyces pyogenes and Pasteurella multocida. Signs of infection did not develop for months in some cattle and were often intermittent. The most common clinical signs included anorexia, lethargy, fever, frontal bone distortion, exophthalmos, abnormal posture, nasal discharge, and neurologic abnormalities. Treatment consisted of trephination at 2 sites, drainage and lavage of the sinus cavity, and administration of antibiotics and analgesics. Eight cattle responded well to treatment and were discharged, but 4 others had signs of CNS involvement and died or were euthanatized. Trephination of the frontal sinus cavity at carefully chosen sites and antibiotic treatment are indicated when sinusitis is suspected. Drainage of the sinus cavity is imperative to avoid extension of the infection into the CNS.


Surgical treatment of colic in American miniature horses: 15 cases (1980-1987).

A study of 15 American miniature horses (AMH) that underwent surgical treatment for colic was performed. Information obtained from the medical records included signalment, clinical signs, type and location of gastrointestinal lesion, and postoperative complications. All 15 AMH had intraluminal obstructions, attributable to feed impactions (11 horses), enteroliths (2), and sand (2). The most common location of obstruction was the small colon, which was involved in 9 of the 15 cases. All 15 AMH survived and were discharged from the hospital. Six of the 15 AMH underwent subsequent surgical treatment for abdominal disorders. Elapsed time between the first and second operations ranged from 1 month to 5 years. Intestinal adhesions were observed in all AMH that were surgically treated twice. Thus, despite the fact that most of the AMH had a simple intraluminal obstruction, 40% (n = 6) developed adhesions that required or complicated a second surgery. Of the 15 AMH, 87% (n = 13) survived at least 12 months after the initial exploratory celiotomy. These findings suggest that most surgical abdominal conditions in AMH can be corrected; however, precautions should be taken to avoid or minimize adhesion formation.


Dorsomedial articular fracture of the proximal aspect of the third metacarpal bone in standardbred racehorses: seven cases (1978-1990).

Seven Standardbred horses, all pacers, with a mean age of 2.9 years (range, 2 to 4 years), had dorsomedial articular fracture of the proximal aspect of the third metacarpal bone. Fracture caused acute, unilateral, severe lameness after training or racing. Lameness was abolished by midcarpal joint anesthesia in 4 horses. Six horses had a palpable bony swelling, which caused signs of pain. Radiography revealed a nondisplaced, articular, oblique fracture extending distad toward the dorsomedial cortex for a mean distance of 28 mm (range, 15 to 40 mm). In all horses, chronic periosteal proliferative changes, seen near the distal aspect of the fracture, corresponded to palpable bony exostoses and were associated with the medial attachment of the extensor carpi radialis tendon. In 1 horse, internal fixation followed by a 6-month rest resulted in a successful outcome. All other horses were given 3 months' rest without surgery and were not lame. Five horses raced successfully and lowered the lifetime race records, 1 horse was sound and trained successfully, but died of colic, and 1 horse was not lame in early training.


Isolation and characterization of new peptide antibiotics, plusbacins A1-A4 and B1-B4.

New antibiotics, plusbacins A1-A4 and B1-B4, were isolated from the culture broth of a strain of Pseudomonas sp. These antibiotics were isolated as a complex by column chromatographies on Diaion HP-20 and silica gel, and then separated by HPLC. They are amphoteric in nature. The hydrochlorides are obtained as colorless powders, soluble in methanol and alkaline water. From their physico-chemical properties, these antibiotics are presumed to be acyloctapeptides containing a lactone linkage, and their differences occur in amino acid and fatty acid residues. The antibiotics are active against Gram-positive bacteria in vitro and in vivo.


Structures of new peptide antibiotics, plusbacins A1-A4 and B1-B4.

The constituent amino acids of plusbacins A1-A4 were determined to be two moles of L-trans-3-hydroxyproline and one mole each of D-threo-beta-hydroxyaspartic acid, L-threo-beta-hydroxyaspartic acid, D-allo-threonine, D-serine, D-alanine and L-arginine. In plusbacins B1-B4, one mole of L-trans-3-hydroxyproline is replaced by L-proline. The fatty acid residue of A1 and B1 was determined to be 3-hydroxy-tetradecanoic acid, for A2 and B2 to be 3-hydroxy-isopentadecanoic acid, for A3 and B3 to be 3-hydroxy-isohexadecanoic acid, and for A4 and B4 to be 3-hydroxy-hexadecanoic acid. A lactone linkage was suggested to reside between L-threo-beta-hydroxyaspartic acid and 3-hydroxy-fatty acid residues by degradation experiments. The amino acid sequences of plusbacins A2 and B2 were confirmed by Edman degradation of their deacylated products, and supported by mass spectrometric studies. From the above, structures of all components of plusbacins were concluded.


Mersacidin, a new antibiotic from Bacillus. Fermentation, isolation, purification and chemical characterization.

Mersacidin (1) is a new peptide antibiotic containing beta-methyllanthionine. It is classified as a member of the proposed lantibiotic group of antibiotics, and is produced by a species of Bacillus. Mersacidin has a molecular weight of 1,824 (C80H120N20O21S4). The antibiotic is active against Gram-positive organisms including methicillin-resistant Staphylococcus aureus, but has no activity against Gram-negative bacteria or fungi.


Mersacidin, a new antibiotic from Bacillus. In vitro and in vivo antibacterial activity.

Mersacidin is a new peptide antibiotic of the proposed lantibiotic family. It is active in vitro and in vivo against Gram-positive bacteria including the methicillin-resistant Staphylococci. Its in vitro activity is less than those of vancomycin and erythromycin but it shows much higher activity in the in vivo system than can be expected from the in vitro testing results. A water soluble potassium salt has been prepared which has an activity profile similar to that of mersacidin, but has better in vivo activity against Streptococcus pyogenes than the parent compound.


SF2457, a new antibiotic related to amicetin.

A novel nucleoside antibiotic, SF2457, was isolated from the fermentation broth of Nocardia brasiliensis SF2457. The structure of SF2457 was determined by degradation studies using alkaline hydrolysis and methanolysis. SF2457 is closely related to the amicetin group antibiotics. The antibiotic exhibited inhibitory activity against Gram-positive and Gram-negative bacteria.


Production and biological activities of a new antifungal antibiotic, TAN-950 A.

A novel antifungal antibiotic, TAN-950 complex, was isolated from the culture filtrate of Streptomyces platensis A-136 (IFO 14603, FERM BP-1786). The water-soluble amphoteric substances in this complex were purified by chromatography using ion-exchange resins, QAE-Sephadex and adsorptive resins and were designated TAN-950 A and TAN-950 A-E mixture. The molecular formula of TAN-950 A was determined to be C6H7N2O4Na for the sodium salt. This new amino acid antibiotic showed antifungal activity against Candida albicans in vitro and in vivo, and had low toxicity in mice.


Sphingofungins A, B, C, and D; a new family of antifungal agents. I. Fermentation, isolation, and biological activity.

In screening for antifungal inhibitors from fungi, four related antifungal agents have been isolated from the cultivation of Aspergillus fumigatus ATCC 20857. These agents were initially produced by the microorganism growing on a solid millet-based medium. A liquid medium containing both glucose and glycerol has also been developed in which these antibiotics are produced in two phases. These novel compounds, sphingofungins A, B, C, and D, show a limited spectrum of antifungal activity but were especially effective against Cryptococcus species.


A new antitumor antibiotic product, demethylchartreusin. Isolation and biological activities.

A new antitumor antibiotic, 3''-demethylchartreusin was isolated from the culture broth of Streptomyces chartreusis, as a minor component of crude chartreusin. It is structurally related to chartreusin, containing same aglycone of chartreusin, but different sugar moieties. 3''-Demethylchartreusin exhibits some potent inhibitory activities against murine tumors.


Depudecin: a novel compound inducing the flat phenotype of NIH3T3 cells doubly transformed by ras- and src-oncogene, produced by Alternaria brassicicola.

A novel compound depudecin inducing the flat phenotype of ras- and src- transformed NIH3T3 cells at a concentration of 1 microgram/ml was isolated from the culture broth of Alternaria brassicicola. Based on its spectroscopic characteristics and X-ray crystallographic analysis of its bis-(1S)-(-)-camphanate, the structure of depudecin was determined to be (2R,3S,4S,5E,7S,8S,9R)-2,9- dihydroxy-3,4;7,8-diepoxy-undeca-5,10-diene.


Adipostatins A and B, new inhibitors of glycerol-3-phosphate dehydrogenase.

As a result of screening for inhibitors of glycerol-3-phosphate dehydrogenase, which may be effective to prevent corpulence, we isolated two inhibitors named adipostatin A and adipostatin B from the culture broth of Streptomyces cyaneus 2299-SV1. Their structures have been established to be 5-n-pentadecylresorcinol and 5-isopentadecylresorcinol, respectively. Adipostatin A and adipostatin B inhibited glycerol-3-phosphate dehydrogenase at the IC50 values of 4.1 microM and 4.5 microM, respectively. These compounds prevented triglyceride accumulation in 3T3-L1 cells at a concentration of the microM level.


PI-200 and PI-201, new platelet aggregation inhibitors produced by Streptomyces sp. A7498. Taxonomy, fermentation, isolation, physico-chemical properties, structure determination and biological properties.

Two new platelet aggregation inhibitors, PI-200 and PI-201 were isolated from the fermentation broth of Streptomyces sp. A7498. PI-200 and PI-201 inhibited ADP-induced aggregation of rabbit platelets with an IC50 of 3.8 x 10(-4) M and 7.1 x 10(-4) M, respectively.


Leualacin, a novel calcium blocker from Hapsidospora irregularis. I. Taxonomy, fermentation, isolation, physico-chemical and biological properties.

A new calcium blocker, designated leualacin, has been isolated from Hapsidospora irregularis. The compound inhibits the binding of 3H-nitrendipine, a well known synthetic calcium blocker, to cardiac Ca channel in a competitive manner, although its structure is completely different from dihydropyridines.


Herboxidiene, a new herbicidal substance from Streptomyces chromofuscus A7847. Taxonomy, fermentation, isolation, physico-chemical and biological properties.

Screening of microbial fermentation broths for herbicidal activity led to the discovery of a novel polyketide, herboxidiene, from an actinomycete identified as a member of the Streptomyces chromofuscus cluster. A 14- to 20-fold increase in fermentation production of herboxidiene was achieved as a result of media optimization. Herboxidiene was purified using successive reverse phase C18 steps and Sephadex LH-20 chromatography. Its molecular formula, C25H42O6, was determined by HRFAB-MS. Herboxidiene demonstrated exceptionally potent, selective, herbicidal activity against a variety of weed species and was inactive against wheat, even at rates as high as 5.6 kg/hectare.


RU 29 246, the active compound of the cephalosporin prodrug-ester HR 916. III. Pharmacokinetic properties and antibacterial activity in vivo.

The pharmacokinetics of the broad spectrum cephem RU 29 246 and its prodrug-ester HR 916 B were investigated in mice, rats and dogs and compared to those of cefpodoxime proxetil, cefuroxime axetil and cefixime. HR 916 B is well absorbed following oral administration and efficiently converted to the antibacterially active form. In mice, mean peak blood levels of 31.1 micrograms/ml of the parent compound were recorded within 20 minutes after oral administration of a single dose equivalent to 40 mg/kg RU 29 246. The bioavailability calculated on the basis of the areas under the concentration-time curves (AUC) and the urinary recoveries was about 90%. In rats, peak blood levels of 14.5 micrograms/ml were obtained 1 hour after an oral 20 mg/kg dose. The bioavailability was calculated as 70%. In dogs, 40% of an oral 10 mg/kg dose was recovered in the urine within 24 hours. Cmax was 15.9 micrograms/ml at 4.6 hours. Mean elimination half-lives of RU 29 246 were 0.35, 0.5 and 2.1 hours in mice, rats and dogs, respectively. After an oral HR 916 B dose equivalent to 50 mg/kg of RU 29 246, tissue concentrations at 0.5 hour ranged between 0.8 micrograms/g in brain and 95.7 micrograms/g in murine kidneys. These values of HR 916 B are similar to, or distinctly higher than, those of the reference compounds. Of the oral cephalosporins tested, HR 916 B had the most balanced antibacterial spectrum. With ED50s of between 0.9 and 11.5 mg/kg against staphylococci, its activity was similar to that of the additional reference compound cefaclor and higher than that of cefuroxime. Cefixime and cefpodoxime proxetil displayed low antistaphylococcal activity or were inactive. In septicemias with Enterobacteriaceae, cefixime and cefpodoxime proxetil were more potent than HR 916 B and cefaclor. Cefuroxime axetil was inactive against most of these infections. HR 916 B was also highly effective against murine lung infections caused by Klebsiella pneumoniae DT-S or Streptococcus pneumoniae 1147.


Studies on cephalosporin antibiotics. V. Synthesis, antibacterial activity and oral absorption of new 3-[(Z)-2-methoxycarbonylvinylthio]-7 beta- [(Z)-2-(2-aminothiazol-4-yl)-2-(oxyimino)acetamido]cephalosporins.

A series of new 3-[(Z)-2-methoxycarbonylvinylthio]-7 beta-[(2- aminothiazol-4-yl)acetamido]-cephalosporins (1) having various oxyimino groups (Z-form) at the alpha position of the C-7 side chain was synthesized and evaluated for antibacterial activity and oral absorption in rats. Of these, the cephalosporin (1a) with a hydroxyimino group in the C-7 side chain showed a potent antibacterial activity against Gram-negative bacteria and Gram-positive Staphylococcus aureus as well as good oral absorption in rats. The structure-activity relationships of 1 are also presented.


Synthesis and in vitro activity of novel quaternary ammonium carbapenems: 2-pyridiniopropyl and 1-pyridinioethyl carbapenems.

The synthesis of new carbapenems having either a pyridiniopropyl group at the 2-position or a pyridinioethyl group at the 1-position is described, along with the preparation of their corresponding hydroxy and acetoxy analogs. The antibacterial activity, susceptibility to dehydropeptidase-I (DHP-I) enzyme and chemical stability of these new carbapenems are also reported. 2-Pyridiniopropyl-carbapenem 4 was found to possess excellent antibacterial activity. It was more stable chemically and less susceptible to the DHP-I enzyme than the thio analog 3. 1-Pyridinioethylcarbapenem 5 showed significantly reduced antibacterial activity as compared to 2-pyridiniopropylcarbapenem 4.


Totally synthetic analogues of siastatin B. I. Optically active 2-acetamidopiperidine derivatives.

Totally synthetic analogues of siastatin B, optically active 2-acetamido-3,4,5-trihydroxypiperidines having the nitromethyl, aminomethyl and carboxyl branched groups at C-5 have been obtained from D-ribono-1,4-lactone by a stereospecific convergent method. Some analogues showed inhibitory activity against some glycosidases.


Totally synthetic analogues of siastatin B. II. Optically active piperidine derivatives having trifluoroacetamide and hydroxyacetamide groups at C-2.

Siastatin B analogues, optically active 2-(trifluoroacetamide)- 3,4,5-trihydroxypiperidines having nitromethyl, aminomethyl and carboxyl branched groups at C-5, and (+)-(2R,3R,4R,5R)-5-(aminomethyl)-3,4,5-trihydroxy-2- (hydroxyacetamido)piperidine have been obtained total synthetically from D-ribono-1,4-lactone. Some analogues have inhibitory activity against some glycosidases, and (+)-(2R,3R,4R,5R)-2-(trifluoroacetamido)-3,4,5-trihydroxypiperi dine-5-carboxylic acid showed a marked inhibitory activity against beta-glucuronidase.


Synthesis and biological properties of 1 beta-methylcarbapenems with N-methylpyrrolidinylthio group at C-2 position.

A series of 1 beta-methylcarbapenem compounds, which have a 5'-substituted-N-methylpyrrolidin-3'-ylthio group as a C-2 side chain, have been prepared and their biological properties were investigated. Substitution with a methyl group on the nitrogen atom in the C-2 side chain effectively enhanced stability to renal dehydropeptidase-I as well as introduction of methylene spacer between the aminocarbonyl group and the pyrrolidine ring of the 5'-aminocarbonylpyrrolidin-3'-ylthio group.


Anthelmintic activity of dioxapyrrolomycin.

Dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 produced by UC 11065 were evaluated as anthelmintics. Assays used to examine these compounds included effects on the free-living nematode Caenorhabditis elegans, ability to clear target nematodes (Haemonchus contortus and Trichostrongylus colubriformis) from jirds, and clearance of Haemonchus contortus from lambs. A crude extract of UC 11065 containing dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 was active against C. elegans and against H. contortus in the jird. Purified and/or synthetic samples of dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 were tested in the jird model; only dioxapyrrolomycin exhibited appreciable activity against H. contortus (greater than or equal to 90.9% clearance at 0.33 mg/jird), while none of the compounds showed appreciable activity against T. colubriformis. Dioxapyrrolomycin cleared 99.9% of H. contortus from lambs at 12.5 mg/kg. An in vitro migration study using susceptible and closantel-resistant H. contortus showed there is cross-resistance between dioxapyrrolomycin and closantel. Dioxapyrrolomycin appears to be a narrow-spectrum anthelmintic which works through a closantel-like mode-of-action.


Neomycin biosynthesis: the incorporation of D-6-deoxy-glucose derivatives and variously labelled glucose into the 2-deoxystreptamine ring. Postulated involvement of 2-deoxyinosose synthase in the biosynthesis.

D-[6-3H3]6-Deoxy-5-ketoglucose (10) and D-[5,6-3H2]6-deoxyglucose (11) were incorporated into neomycins B and C using a growing culture of Streptomyces fradiae. D-[6-3H]6-Deoxy-5-ketoglucose was incorporated into neomycin, as efficiently as the well established precursor D-glucose, and was found to label exclusively the 2-deoxystreptamine ring of the antibiotic. The results strengthened the previous proposals that in the formation of 2-deoxystreptamine the C-6 hydroxyl group of D-glucose is removed prior to the cyclisation reaction. Studies using the incorporation of D-[3-3H]glucose, D-[3,4-3H2]glucose and D-[5-3H]glucose into neomycin followed by the degradation of the latter established that in the biosynthesis of the 2-deoxystreptamine ring the C-4 and C-5 hydrogen atoms of glucose are removed. The loss of the C-4 hydrogen atom of the glucose is attributed to the formation of a 4-keto derivative which facilitates the removal of the C-5 hydrogen atom thus setting the stage for the expulsion of the C-6 hydroxyl group. The 5,6-olefinic intermediate formed in the process then undergoes cyclisation eventually releasing 2-deoxyinosose. The enzyme systems which participate in the conversion of D-glucose equivalent into 2-deoxyinosose may be described as 2-deoxyinosose synthase that in broad mechanistic terms resembles dehydroquinate synthase.


Two-tone suppression in inner hair cell responses: correlates of rate suppression in the auditory nerve.

Inner hair cell (IHC) recordings were made from second turn of the guinea pig cochlea where characteristic frequencies are approximately 4000 Hz. In order to compare IHC responses with rate suppression measured in the auditory nerve, suppressors were introduced that produced little or no response in the hair cell. The effects of a variable-frequency suppressor on a constant-frequency probe, placed near characteristic frequency, were also investigated since this paradigm is commonly used in single unit experiments. Resulting magnitude changes were measured in the fundamental component of the ac receptor potential and/or in the total dc produced in the region of temporal overlap between the two stimulus inputs. This latter component is especially important when considering how changes in IHC responses relate to decreases in discharge rate in single auditory nerve fibers. Since the ac receptor potential is filtered by the hair cell's basolateral membrane, the dc component probably controls transmitter release at the characteristic frequency of these second-turn IHCs. Based on results from these and previous experiments, a proposal is advanced to explain the evolution of two-tone suppression in the peripheral auditory system. The paper also discusses the use of excitatory versus non-excitatory suppressors and includes a description of two-tone suppression areas at the mechanical, IHC and single unit levels. The explanation of low-side suppression areas is of special interest since hitherto they have been difficult to model (Kim, 1985).


Metabolic labelling and quantitation of proteins synthesized by single chick cochleas.

Molecular studies of the peripheral auditory system are made difficult by the small quantities of tissue available and by their relative inaccessibility. In addition, the cochlea and other hair cell-containing receptor organs are composed of both hair cells and supporting cells, as well as several other cell types. The identification of known proteins and the characterization of specific and novel protein molecules from these tissues require the use of sensitive techniques and a consideration of the complex histology. The chick cochlea was selected as an experimental system since the cochlea is relatively accessible in the bird, the receptor neuroepithelium contains a large number of hair cells compacted in a small area, and the physiology of the auditory periphery has been studied extensively. A general procedure is described for the metabolic radiolabelling of proteins from single cochleas followed by their solubilization, separation by high-resolution two-dimensional gel electrophoresis, and accurate quantitation. The method is highly reproducible and sensitive, and should prove useful in studies of proteins from the specialized cell types of the chick cochlea, including the identification of those whose rates of synthesis are modified in response to acoustic stimulation and sound damage or recovery.


Cochlear efferent neurons projecting to both ears in the chicken, Gallus domesticus.

Different retrograde neuroanatomical tracers were injected into each cochlea of adult chicken. The number of cells labeled in the cochlear efferent cell group found bilaterally within the caudal pontine reticular formation depended upon the tracer, with True Blue and Fluoro Gold yielding maximal average counts of 332 efferent neurons per injection. Double labeling of less than 1% of these cells was possible with the combination of True Blue and Diamidino Yellow. Thus the contribution of efferent neurons with axon collaterals projecting to both ears is not fundamentally different in birds and other vertebrates.


C-type potassium channels in the lateral cell membrane of guinea-pig outer hair cells.

The basolateral cell membrane of outer hair cells (OHC) from the mammalian cochlea is known to contain K(+)-channels. The prevailing type, a high-conductance K(+)-channel was further characterized in the present study in order to support its classification as C channel. OHC were isolated from the 3rd and 4th turn of the guinea-pig cochlea. Cell-attached and excised inside-out patches of the lateral cell membrane were investigated. The C-type channel had a selectivity for K+ over Na+ of 12:1 to 20:1 and displayed Goldman-type rectification and voltage-dependence of the open probability. The kinetics of both opening and closing could be described by time constants in the range of ms. The channel provides a calcium- and voltage-activated pathway through OHC lateral membranes for passive K+ transport.


Low-frequency modulation of compound action potential in experimental perilymphatic fistula and endolymphatic hydrops.

We have tested the hypothesis that the cause of cochlear dysfunction associated with perilymphatic fistula (PLF) is closely related to endolymphatic hydrops (ELH). Using guinea pigs, we studied the tone-burst elicited compound action potential (CAP) and its modulation as caused by a 50 Hz biasing tone in experimental PLF. We compared these results with those of experimental ELH. Following perilymph aspiration through the perforated round window membrane, mild but significant elevations of CAP thresholds at tested frequencies were found. A reduction in the amplitude of cochlear microphonics (CM) for a 50 Hz sine wave appeared to correlate with these CAP threshold changes. However, there were no significant changes in the modulation effect of the 50 Hz biasing tone on the CAP elicited by an 8 kHz tone burst. This finding differed from that in ears with experimental ELH, in which significant reductions of both 50 Hz CM and the degree of CAP modulation were consistently observed. We concluded that it is unlikely that the underlying mechanisms of a modification to the low frequency response of the base of the cochlea following perilymph aspiration is linked to that of experimental ELH.


Morphology of HRP-labelled cochlear nerve axons in the dorsal cochlear nucleus of the developing hamster.

To study the development of the central terminal arbors of the cochlear nerve fibers in the dorsal cochlear nucleus, horseradish peroxidase-labelled axons in young and adult hamsters were analyzed morphometrically. Brainstem slices with whole cochlear nuclei were maintained in a slice chamber and the cochlear nerve root was injected with a mixture of wheat germ agglutinin-horseradish peroxidase, horseradish peroxidase and poly-L-ornithine. The poly-L-ornithine was added to keep the injection site small; small injections resulted in only a few axons being labelled and permitted reconstruction of individual fibers. Axons underwent an initial period of ingrowth that was completed prior to the onset of hearing (postnatal day 16). After this time the morphology and area of influence of the axons remained unchanged but the nucleus continued to increase in size. Since no additional cochlear nerve axons grow into the nucleus during this period of nuclear growth, the existing axons necessarily become more widely spaced as development proceeds. These anatomical changes may contribute to the progressive narrowing of auditory cell tuning curves.


Quantitative carbohydrate analyses of the tectorial and otoconial membranes of the guinea pig.

Carbohydrate composition of the tectorial membrane (TM) and the otoconial membrane (OM) of the guinea pig was analyzed after hydrolysis, using high-performance anion-exchange chromatography and pulsed amperometric detection. Both of the tissues were highly glycosylated; the carbohydrate content being 24-42% of protein. GlcN, Gal, Glc and Man were found to be the major component sugars of TM, whereas little GalN was found. Fuc and NANA were also present, but NGNA was not detectable. After digestion with thermolysin for solubilization, OM was separated into two fractions: insoluble mineral particles of the otoconia (OM-ppt) and a soluble fraction from the gelatinous layer (OM-sup). These two fractions showed distinct carbohydrate composition from each other. Further analyses using glycosidases revealed that TM contained asialyl and monosialyl but little di-, tri- and tetrasialyl N-glycosides, and OM-sup did not seem to be susceptible to endo-beta-galactosidase, which is known to cleave some N-acetyl-polylactosamine and keratan sulfate. Based on these analyses, it can be suggested that most of the carbohydrates in TM are likely to be asialyl and monosialyl N-glycosides. N-Glycosides may be predominant in the otoconia as well, and a polymer structure consisting of GlcN(Ac) and Gal other than N-acetyl-polylactosamine may exist in the gelatinous layer of OM. O-Glycosylation of the usual type appeared to be minor in all the fractions.


Comparisons of the development of auditory brainstem response latencies between cats and humans.

Developmental changes in the peak latencies of auditory brainstem responses (ABR) recorded from humans and kittens were compared to test the hypothesis that developmental time courses among mammals are the same when appropriately normalized. Response latencies were computed as the difference from adult latency and conceptional ages were represented as percentages relative to the age that ABR wave latencies achieved a criterion value within 0.2 ms of asymptotic latency (i.e., adulthood). An underlying assumption of this exercise is that far-field response latency is an appropriate index of overall 'auditory development'. Results of this analysis suggest that developmental changes in latency of responses arising within the auditory periphery are similar between humans and cats, when appropriately normalized, and that more central changes show less correspondence. Consequently, absolute time course differences for specific developmental parameters must be considered and caution should be exercised when extrapolating results acquired from one species to the other.


Rate and frequency interactions in the auditory brainstem response of the adult rat.

Tone pip stimuli were used at different repetition rates to examine influences upon auditory brainstem response components. Rate increases to 80.1/s result in significant latency increases in waves IV and V at all test frequencies. Rate effects occur in rostral brainstem response components which show frequency related latencies different from wave I. Amplitude measures decline at higher repetition rates for waves I and IV at most test frequencies. Rate effects are most pronounced at 8 kHz stimulation which is around the optimal behavioral frequency in rat. At higher rates the second peak of wave I is enhanced at 8 kHz, whereas the third peak of wave I at 40 kHz disappears. These results demonstrate that tone-specific stimuli provide sensitive measures of latency, amplitude and wave morphology of components of the auditory brainstem response as a function of stimulus repetition.


Distribution of F-actin and fodrin in the hair cells of the guinea pig cochlea as revealed by confocal fluorescence microscopy.

We double-stained paraformaldehyde fixed guinea pig cochleas with rhodaminated phalloidin to detect F-actin and with a monoclonal antibody against non-erythroid spectrin (fodrin). The hair cells were studied in surface specimens of the organ of Corti with confocal fluorescence microscopy. In serial optical sections, phalloidin stained the stereocilia, cuticular plate, and a circumferential ring beneath it in the inner and outer hair cells (IHCs and OHCs). The cytoplasm of the IHCs and the OHCs was unlabelled, but the infracuticular network of the OHCs in the upper turns showed a strong reaction. The lateral plasma membrane was unreactive with phalloidin in the IHCs and OHCs, except in the basal turn, where a moderate reaction, probably representing actin of Deiter's cups, was seen along the lateral walls of the basal pole of the OHCs. Fodrin was similarly seen in the cuticular plate, in a circumferential ring beneath it, and in the infracuticular network of the apical OHCs. The most interesting finding was the fodrin-specific distinct labelling of the lateral cell surface in the OHCs of the basal cochlear turn. This staining diminished towards the apex and was practically absent in the OHCs located above the level of 15 mm from the round window. The lateral cell surface of IHCs showed moderate fodrin labelling in all cochlear turns. This staining was much weaker than that seen in the basal OHCs. Fodrin labelling revealed deformation from the regular cylindrical shape in midportion of the OHC bodies in the basal turn of the cochlea.


Forward and reverse transduction at the limit of sensitivity studied by correlating electrical and mechanical fluctuations in frog saccular hair cells.

The spontaneous fluctuations of the intracellular voltage and the position of the sensory hairbundle were measured concurrently using intracellular microelectrodes and an optical differential micro interferometer. Magnitude and frequency distribution of the hair bundles' spontaneous motion suggest that it consists mostly of Brownian motion. The electrical noise, however, exceeds the value expected for thermal Johnson noise by several orders of magnitude, and its frequency distribution reflects the transduction tuning properties of the hair cells. Frequently, a strong correlation was observed between the fluctuations of the hair bundle position and the intracellular electrical noise. From the properties of the correlation and from experiments involving mechanical stimulation we conclude that in most cases mechano-electrical transduction of the bundles' Brownian motion causes this correlation. Small signal transduction sensitivities ranged from 18 to 500 microV/nm. Bundle motion that was observed in response to current injection in more than half of the cells suggests the existence of a fast reverse (electro-mechanical) transduction mechanism to be common in these cells. The sensitivities could be as high as 600 pm of bundle deflection per millivolt of membrane potential change. In a significant minority (4 in 44) of cells, all showing excess electrical noise, we found 'non-causal' components of the electro-mechanical correlation, and in two of those cells narrow-band bundle motion in excess of their thermal motion at frequencies coincident with peaks in the intracellular noise was observed.


The epidemiology of ovarian cancer.

The epidemiologic data collected to date have provided some important and provocative clues as to the etiology of ovarian cancer. The recognition of familial clustering of this disease has led to exciting advances in understanding the genetics involved. The contribution of endocrine factors has been well documented in the epidemiologic literature, leading to important insights into the process of carcinogenesis. Clearly, additional information is needed to further explain the interplay of genetic, physiologic, and life style factors, to lead to a better understanding of the disease, and ultimately to a means of prevention and control.


Pathology of epithelial tumors.

The pathology of epithelial tumors is reviewed in this article, with particular emphasis on new information. Important new data of prognostic significance concerning tumors in the borderline group are emphasized, and the division of mucinous borderline tumors into two groups is discussed. Serous carcinomas of surface type are reviewed, which have recently been the subject of a great deal of study, as well as transitional cell carcinoma, a newly defined subtype of invasive cancer. Finally, the highly aggressive small cell carcinoma is briefly discussed.


Prognostic factors in ovarian cancer.

Epithelial ovarian cancers vary considerably in their biologic behavior and this is reflected in the variety of clinicopathologic factors that are used for predicting outcome. This article assesses the potential value of some of the newer prognostic factors and critically evaluates the more commonly used clinicopathologic variables.


Hereditary ovarian carcinoma.

No woman is at greater risk for ovarian carcinoma than one who is a member of a hereditary ovarian carcinoma syndrome kindred and whose mother, sister, or daughter has been affected with this disease and with an integrally related hereditary syndrome cancer. This article surveys the existing understanding about hereditary ovarian carcinoma. Emphasis is given to its diagnosis, heterogeneity, interpretation, and the application of this information to improving cancer control.


Oncogenes in ovarian cancer.

The discovery of cancer-causing genes has provided us with the exciting opportunity to begin to understand the molecular pathology of ovarian cancer. Activation of several of these genes including HER-2/neu, myc, ras, and p53 has been described in some ovarian cancers (Table 2). In addition, some proto-oncogenes such as the EGF receptor (erbB) and the M-CSF receptor (fms) are expressed along with their respective ligands in some ovarian cancers. Finally, for every oncogene that has been studied in ovarian cancer, there are at least a half-dozen that remain unexplored. In the future, when we have a better understanding of the molecular pathology involved in the development of ovarian cancer, this may allow us to better diagnose and treat, and eventually prevent, ovarian cancer.


Growth regulation of ovarian cancer.

The disease referred to as ovarian cancer is composed of those tumors derived from the ovarian surface epithelium. Ovarian cancer incidence peaks in postmenopausal women, when the hormonal milieu is in a state of flux. The ovarian carcinomas have histologic characteristics of classical endocrine responsive tissues. For example, the serous tumors are similar in appearance to the epithelium of the fallopian tube, the mucinous tumors to that of the endocervix, and the endometrioid tumors to that of the endometrium. These observations alone suggest a role for hormones in disease etiology and progression. In addition, much experimental evidence has accumulated that shows direct hormonal and growth factor effects on the normal and neoplastic ovarian surface epithelium and the potential for responsiveness based on the presence of specific receptors. The concept that growth stimulatory substances, by their impact on mitosis and cell number, may influence the rate of mutations that could confer malignant transformation serves as an additional mechanism by which growth stimulatory substances can influence ovarian cancer initiation. With the emergence of new molecular biology techniques, future studies should unravel the mechanisms by which steroid hormones, peptide hormones, and peptide growth factors influence the development of ovarian cancer.


Ovarian cancer. Early diagnosis and screening.

Late diagnosis is the main reason for the poor outcome of ovarian cancer patients. An attempt to diagnose the disease methods for early diagnosis was recently investigated. The main effort of the study was in finding the role of ultrasonography and CA-125 and their sensitivity and specificity in the screening procedure. The preliminary reports suggest that a mortality study with a large women population has to be done before the usefulness of screening these methods can be established.


Management of early-stage ovarian cancer.

Approximately 30% of patients with epithelial ovarian cancer present with localized or early-stage disease. Accuracy of staging is dependent on careful surgical staging. Subsets of patients with specific prognostic factors have now been identified who do not require additional therapy after proper primary surgical staging. Poor prognosis patients do require adjuvant therapy. Whole abdominal irradiation, intraperitoneal P32, and single agent and combination chemotherapy have been utilized. The optimal therapy or whether any therapy is truly effective remains unknown.


Chemotherapy for advanced epithelial ovarian cancer.

In the past decade there have been significant improvements in chemotherapy for advanced ovarian cancer. Platinum-based chemotherapy has improved response rates and, to a lesser degree, prolonged survival. Taxol and its combination with platinum drugs holds the promise for even further improvements in survival. Clinical trials are evaluating issues such as dose-intensity, new combinations, and drug resistance.


The current role of radiotherapy in the management of ovarian cancer.

The role of radiation in the management of ovarian carcinoma continues to be a controversial issue. This article discusses some of the controversies surrounding treatment with radiotherapy in the context of an analysis of the reasons that have led to the apparent failure of radiotherapy to cure many patients and describes several new experimental strategies with a potential for future improvement. Although the recent outcome of the combined modality approach has been extremely frustrating, the prospects for future developments of the experimental leads described in this article are encouraging and will hopefully translate into more effective results of this approach in ovarian carcinoma.


Intraperitoneal therapy in the management of patients with ovarian cancer.

Basic pharmacologic principles support the concept that more drugs can be delivered to the tumor when it is delivered through the intraperitoneal route. Pharmacokinetic studies have confirmed that for many chemotherapeutic agents a substantial pharmacologic advantage can be achieved using this approach. Preliminary studies have suggested that intraperitoneal chemotherapy may be especially advantageous in patients with small-volume disease or in patients who have a negative second-look laparotomy. Clinical studies are ongoing to further define the role intraperitoneal therapy will play in the management of patients with ovarian cancer.


Ovarian cancer. Experimental chemotherapy.

Ovarian cancer is a drug-sensitive disease, yet most patients still succumb after initial response. Experimental approaches to this disease include the discovery of new drugs, including taxol and ifosfamide; evaluation of candidate drugs such as tetraplatin and topotecan; and approaches to overcome intrinsic and/or acquired resistance. The latter has been addressed by increasing dose-intensity of currently available therapy, developing agents to abrogate dose-limiting toxicity, and by attempts to interfere with mechanisms of resistance.


Biologic and immunologic therapy of ovarian cancer.

Conventional cytotoxic chemotherapy fails to cure the majority of patients with advanced-stage ovarian cancer, in spite of encouraging initial antitumor responses. With the emergence of drug resistance in refractory tumors, new biologic and immunologic treatment strategies are needed. Small-volume residual peritoneal disease remains an attractive target for therapeutic trials; however, even in this optimal circumstance, few regimens have yet achieved a high frequency of pathologically confirmed complete remissions. Considerable progress has been made in understanding the impact of growth factors and their receptors on tumor growth regulation and modulation of response to chemotherapy. Better characterization of the antigens recognized by monoclonal antibodies, as well as sequencing of the antibodies themselves, has permitted the rational design of therapeutic reagents that take full advantage of molecular biology techniques for production and conjugation. Important limitations of preclinical models for prediction of host toxicity are recognized, and the reasons for treatment failure in situ, as well as strategies to prevent serious dose-limiting toxicities, are being explored. Further developments in cytokine biology, adoptive cellular therapy, monoclonal antibody conjugation, and molecular biology will continue to provide a growing array of reagents for critical evaluation.


Epinephrine and calcium have similar oxygen costs of contractility.

We compared the oxygen cost of increasing ventricular contractility using Emax (slope of the ventricular end-systolic pressure-volume relation) as the index of ventricular contractility. Contractility was enhanced by calcium and epinephrine in paired experiments on dog left ventricles. Firstly, we obtained left ventricular oxygen consumption (VO2) and systolic pressure-volume area (PVA, a measure of total mechanical energy) of contractions at different volumes in the control contractile state to determine a reference VO2-PVA relation. PVA was obtained as the area in the pressure-volume (P-V) diagram which was bounded by the end-systolic P-V line, end-diastolic P-V curve and systolic P-V trajectory of individual contractions. Secondly, we gradually enhanced Emax with calcium and epinephrine in two consecutive runs at a fixed ventricular volume. Both VO2 and PVA increased with enhanced Emax. From these VO2-PVA data, we calculated the PVA-independent VO2 values at the respective enhanced Emax levels and determined the oxygen cost of Emax as the slope of the relation between the PVA-independent VO2 and Emax. The cost per beat and per 100 g was 0.00158 ml O2/(mmHg/ml) for calcium and 0.00166 ml O2/(mmHg/ml) for epinephrine on average, values not significantly different from each other (P less than 0.05). We conclude that epinephrine and calcium have similar oxygen costs of contractility over a wide range of Emax despite their different pharmacological mechanisms of positive inotropism.


Effect of flosequinan on exercise capacity and cardiac function in patients with chronic mild heart failure: a double-blind placebo-controlled study.

Although beneficial effects of a new vasodilating agent, flosequinan, have been demonstrated in patients with severe heart failure, its efficacy has not been studied in patients with a less severe form of chronic heart failure. In this study, the effects of 4 weeks' administration of flosequinan, 50 mg daily, and placebo on exercise capacity, cardiac function, and symptoms of heart failure were investigated in 24 patients with chronic mild heart failure (New York Heart Association functional class, mainly class II) in a double-blind clinical trial. When the parameter changes during the treatment period of the flosequinan and placebo groups were compared, no significant difference was found in any of the measurements except for left ventricular fractional shortening determined from M-mode echocardiograms; it was increased by 2.9 +/- 1.3% in the flosequinan group whereas it was decreased by 1.3 +/- 0.9% in the placebo group (P less than 0.05 vs flosequinan treatment). However, when compared to baseline values, flosequinan significantly increased exercise time in the symptom-limited maximal exercise test (704 +/- 103 to 763 +/- 107 s, P less than 0.05) and the oxygen uptake at the anaerobic threshold (13.8 +/- 1.3 to 16.7 +/- 1.4 ml/min kg, P less than 0.05), and improved symptoms assessed with a new heart failure severity classification (a median value of 2.0-1.5, P less than 0.05). These improvements were not observed in the placebo group. Serious adverse effects were not observed in either group. These results suggest that flosequinan is useful for the treatment of chronic mild heart failure as well as severe heart failure.


Reverse redistribution on Tl-201 SPECT images after reperfusion therapy for acute myocardial infarction: possible mechanism and prognostic implications.

So-called reverse redistribution on stress Tl-201 imaging has been reported previously, but its significance and clinical implications are not well understood. In patients who received reperfusion therapy for acute myocardial infarction, we frequently observed reverse redistribution on stress Tl-201 images. To investigate the significance of reverse redistribution, 61 patients who underwent reperfusion within 4 h of the onset of chest pain underwent submaximal exercise Tl-201 imaging 3 weeks later. We performed simultaneous coronary arteriography and left ventriculography. We divided these 61 patients into three groups based on the pattern of Tl-201 images. Reverse redistribution was found in 19 patients (Group A), 12 patients had redistribution (Group B), and 30 patients had nonreversible defects (Group C). All patients in Group A had less residual stenosis than those in the other groups, and showed significant improvement of left ventricular function. Furthermore, 12 patients (Group A) demonstrated reverse redistribution or a normal pattern in a follow-up study performed 12 months later. However, in the delayed images the defect was smaller than that shown in the previous study. None of the patients had any symptoms and all returned to their previous occupations. In conclusion, reverse redistribution was common in patients undergoing reperfusion therapy for acute myocardial infarction, especially those with little residual stenosis. Reverse redistribution appears to indicate improved regional wall function in such patients.


Morphological and functional study of free arterial grafts.

Morphological and functional changes of free arterial grafts in dogs were studied for 3 weeks after implantation and the changes were compared to those in implanted free vein grafts. In the arterial grafts, endothelial cells with abundant pinocytotic vesicles and some cytoplasmic folds were observed by transmission and scanning electron microscope and cell detachment was seen only at the site of anastomosis, while most cells were detached in the vein grafts. The site of mechanical damage in the arterial grafts was covered by regenerated endothelial cells which showed similar morphological findings to the normal arterial endothelial cells. In contrast, regenerated cells in the vein grafts started to cover the denuded area 7 days after the implantation and had completely covered it by 3 weeks. Prostacyclin was produced more abundantly in arterial grafts than in vein grafts at any phase after implantation. The level of prostacyclin production was between 30 and 40 pg/mg in any phase after implantation of free arterial grafts, while in vein grafts the level was 2.5 pg/mg at the day of implantation and increased to 13.6 pg/mg at 21 days. This study showed that the endothelial cells were well preserved and the level of prostacyclin production was high in the arterial grafts, and thus the grafts seemed to show potent anti-thrombogenicity after implantation. Although late changes in arterial and vein grafts were not investigated in this experimental protocol, these results may suggest that the arterial graft is superior to the vein graft even in the early period after its implantation as a free graft.


Papillary fibroelastoma in the left ventricular outflow tract.

We report a case of a papillary fibroelastoma originating from the left ventricular endocardium in the outflow tract which was discovered by echocardiography in an asymptomatic patient. Two echocardiographic features were observed: (1) the tumor surface was smooth, and characteristic papillary formation was not detected; and (2) the outline of the mass was clearly defined as a dense echo, with the central, radiolucent, portion surrounded by a highly refractive linear echo at the level of the maximum diameter of the mass. The excised tumor was covered with a gelatinous substance that masked multiple papillae on the surface, but its echolucent center could not be explained by the pathology of the tumor which was solid centrally. Our case indicates that a papillary fibroelastoma may sometimes show echocardiographic findings similar to those of a myxoma, although other investigators have not noted the smooth surface and the echolucent center makes it indistinguishable from a myxoma. Thus, in some cases, it is difficult to distinguish papillary fibroelastoma from myxoma by echocardiography.


Poor response to fluoxetine: underlying depression, serotonergic overstimulation, or a "therapeutic window"?

Symptoms of serotonergic overstimulation may resemble depressive symptoms. Postulating that overmedication with fluoxetine can appear as response failure (as norfluoxetine accumulates), systematic trials of lower doses were conducted in patients who failed to respond despite apparent initial improvements. Of 23 consecutive outpatients treated with fluoxetine 20 mg/day for DSM-III-R major depression, 4 failed to sustain initial improvements during 4-8 weeks of treatment (in the absence of apparent side effects). In these 4 patients, fluoxetine was withdrawn for 2 weeks, then reinstituted at 20 mg q.o.d. All patients were followed up weekly to monthly (for up to 17 months) and administered the 17-item Hamilton Rating Scale for Depression. Four of 4 patients improved during washout and went on to respond to the lower dose. All 4 cases are presented. On review of the literature, fluoxetine fixed-dose studies reveal increased adverse effects with no increase in efficacy at dosages above 5 mg/day and decreased efficacy at dosages above 40 mg/day. Special issues inherent in the study and use of an antidepressant with a 1- to 3-week active half-life are discussed. Even in the apparent absence of side effects, nonresponse to fluoxetine may be due to overmedication in some patients. Standard doses of fluoxetine may be higher than "optimum." The apparent difficulty distinguishing fluoxetine's adverse effects/toxicity (or a "therapeutic window" effect) from underlying depressive symptoms, taken in conjunction with the 3-9 weeks required to approach steady state, may suggest the option of lowering the dose in some cases of nonresponse or "relapse."


Does fluoxetine exacerbate Parkinson's disease?

Because fluoxetine may be associated with an induction or exacerbation of parkinsonism, caution has been suggested when considering fluoxetine as an antidepressant for patients with Parkinson's disease. We retrospectively reviewed the medical records of 23 outpatients with Parkinson's disease who were receiving or had received fluoxetine. One author evaluated all patients using the Northwestern University Disability Scale for scoring parkinsonism. Rather than employing a formal depression scale, we assessed depression globally. Concurrent medications were permitted. Twenty of the 23 patients experienced no worsening of parkinsonism while being treated with up to 40 mg of fluoxetine per day. The other 3 patients' parkinsonism worsened to a mild degree: a 74-year-old man experienced an increase in akinesia, tremor, and rigidity; a 77-year-old man experienced a slight worsening in tremor and rigidity; and a 56-year-old man experienced a decline in gait and akinesia. It was unclear if these declines, which were neither acute nor severe, were due to fluoxetine treatment or the progression of the disease. Signs of parkinsonism in 2 patients appeared to improve during fluoxetine treatment. Fluoxetine, in doses up to 40 mg/day, does not appear to be associated with exacerbations of parkinsonian signs and symptoms in outpatients with Parkinson's disease. Further investigation of fluoxetine for the treatment of depression in patients with Parkinson's disease is warranted.


DSM-III-R brief reactive psychosis among Air Force recruits.

Clinical characteristics of patients diagnosed with brief reactive psychosis based on DSM-III-R criteria and the incidence of this disorder in a defined population have not been sufficiently studied. Some military recruits who are subjected to relatively uniform levels of stress in a monitored environment over a specified time period develop transient, severe psychotic symptoms consistent with a diagnosis of brief reactive psychosis. We retrospectively reviewed all cases of brief reactive psychosis that occurred among 139,360 Air Force recruits during 6-week basic training experiences over a 3-year period. Brief case descriptions, including clinical courses, are provided. Six cases were found among 557 consecutive psychiatric admissions between January 1, 1988, and January 1, 1991 (annual incidence = 1.43 cases per 100,000 recruits). An apparent temporal relationship was found between the onset of the stressor (basic military training) and the development of psychosis. Paranoid symptomatology, including persecutory delusions, was present in all cases. Adjustment disorders were the most common diagnoses in patients admitted from the training environment (N = 464). Brief reactive psychosis is a rare discharge diagnosis in this large population of young Air Force recruits. Prospective studies are warranted to evaluate the usefulness of DSM-III-R criteria for this disorder and to determine the longitudinal course of patients who have received this diagnosis.


Premenstrual exacerbation of depression: one process or two?

Premenstrual symptoms occur in the setting of other psychiatric disorders, particularly affective disorders. The nosologic issue of whether premenstrual syndrome is an entity distinct from other psychiatric disorders is controversial. We review the association between depression and premenstrual syndrome and describe symptoms in a small series of patients (N = 5) with premenstrual syndrome both during and after resolution of major depression. The overall symptom severity decreased after antidepressant treatment, but this decrease was only significant for dysphoria. In two subjects, other cyclical symptoms consistent with premenstrual syndrome became more apparent after treatment of major depression, possibly because background symptoms of depression improved. Premenstrual symptoms, including dysphoric changes and irritability, can continue despite effective treatment of major depression.


Establishment of anti-human cholesteryl ester transfer protein monoclonal antibodies and radioimmunoassaying of the level of cholesteryl ester transfer protein in human plasma.

Plasma cholesteryl ester transfer protein (CETP) facilitates the net transfer and exchange of cholesteryl ester (CE), triglyceride (TG), and phospholipids between lipoproteins. A series of monoclonal antibodies (mAbs) against human CETP was obtained, comprising mAbs either inhibiting or not inhibiting these transfer activities. One mAb (LT-J1) inhibited the transfer activity of TG almost completely, but not that of CE, indicating that CE and TG binding sites on the CETP molecule may be distinct from each other, and that this mAb may specifically recognize the TG binding site. A radioimmunoassay system for determining the level of CETP was also established using these mAbs, and the plasma CETP levels in 20 normolipemic Japanese adults were found to range from 2.1 to 2.7 mg/liter.


Resonance Raman study on complexes of medium-chain acyl-CoA dehydrogenase.

Resonance Raman (RR) spectra of the complex of pig kidney medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA and of the purple complex formed upon the addition of octanoyl-CoA to the dehydrogenase were obtained. RR spectra were also measured for the complexes prepared by using isotopically labeled compounds, i.e., [3-13C]-, [1,3-13C]-, and [2,4-13C2]acetoacetyl-CoA; [1-13C]octanoyl-CoA; the dehydrogenase reconstituted with [4a-13C]- and [4,10a-13C2]FAD. Both bands of oxidized flavin and acetoacetyl-CoA were resonance-enhanced in the 632.8 nm excited spectra of the acetoacetyl-CoA complex; this confirms that the broad long-wavelength absorption band is a charge-transfer absorption band between oxidized flavin and acetoacetyl-CoA. The 1,622 cm-1 band was assigned to the C(3)=O stretching mode coupling with the C(2)-H bending mode of the enolate form of acetoacetyl-CoA and the bands at 1,483 and 1,119 cm-1 were assigned to bands associated with the C(2)=C(1)-O- moiety. Both bands of fully reduced flavin and the substrate were resonance-enhanced in the 632.8 nm excited spectra of the purple complex. As the enzyme is already reduced, the substrate must be oxidized to octenoyl-CoA; the complex is a charge-transfer complex between the reduced enzyme and octenoyl-CoA. The low frequency value of the 1,577 cm-1 band, which is associated with the C(2)-C(1)=O moiety of the octenoyl-CoA, suggests that the enzyme-bound octenoyl-CoA has an appreciable contribution of C(2)=C(1)-O-.(ABSTRACT TRUNCATED AT 250 WORDS)


A novel ceramide trihexoside from the eggs of the sea urchin, Hemicentrotus pulcherrimus.

Glucosylceramide (Glc beta 1-1Cer) and a novel ceramide trihexoside (Gal beta 1-6Gal beta 1-6Glc beta 1-1Cer) were purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, chromic acid oxidation, enzymatic hydrolysis, enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ceramide trihexoside has a novel carbohydrate structure, and its core structure, Gal beta 1-6Glc, is also novel. The ceramide moieties of these glycolipids are almost identical. Two fatty acids, 22:1 and 22h:1, constitute more than 80% of the total acids. Long-chain bases are all phytosphingosine, approximately 90% of which is n-t18:0. The finding of melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) from the eggs of another sea urchin species [Kubo, H. et al. (1988) J. Biochem. 104, 755-760] and the present finding of the novel ceramide trihexoside suggest that there are a variety of unique sugar structures in sea urchin glycosphingolipids.


Structure-activity relationship in buffalo spleen cathepsin B.

Effect of pH, urea, and guanidine hydrochloride on the activity and structure of buffalo spleen cathepsin B was investigated. At alkaline pH, there was an irreversible loss of the structure as well as the activity of the buffalo enzyme. At acidic pH, however, the inactivation of the enzyme was reversible. The enzyme reversibly lost most of its activity at denaturant concentrations which did not cause a significant change in its secondary structure. The inactivation could be attributed to minor perturbations in the environment of the amino acid residue(s) at and/or around the active site of the enzyme. High urea/guanidine hydrochloride concentrations leading to the structural changes in cathepsin B made the inactivation process irreversible.


Developmental and liver-specific expression directed by the serum amyloid P component promoter in transgenic mice.

Transgenic mice were produced by microinjection of a human serum amyloid P component (hSAP) gene or a fusion gene (SS) comprising the promoter for hSAP (nucleotides -600 to -14 from the start codon) and the coding region of the hepatitis B virus surface antigen (HBsAg). In adult mice, both transgenes were expressed only in the liver, and thus the pattern of expression resembled that of the endogenous mouse SAP gene. Both hSAP mRNA and HBsAg were first detected in liver on the second postnatal day. The level of these products increased rapidly and reached the maximum within the first week. These results suggest that the hSAP gene contains a short, cis-acting, developmental, and liver-specific regulatory sequence at the 5' or the 3' end and that this sequence can target expression of the foreign gene.


Purification and characterization of an 85 kDa sialoglycoprotein in rat liver lysosomal membranes.

Sialoglycoprotein with a molecular mass of 85 kDa (LGP85) was purified from rat liver lysosomal membranes with a 0.9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included: preparation of lysosomal membranes, elimination of LGP107 and LGP96 with immunoaffinity columns, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. LGP85 contains about 22.8% carbohydrate and the carbohydrate moiety is composed of mannose, galactose, fucose, glucosamine, galactosamine, and neuraminic acid, in a molar ratio of 40:20:2:23:3:13. Susceptibility to neuraminidase and immunoreactivity of the protein in intact tritosomes were examined to study the topology of the protein in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the protein were not observed in intact tritosomes until the tritosomes had been disrupted by osmotic shock. These observations suggest that both oligosaccharide chains and the main protein portion of the protein are located on the interior surface of the tritosomal membranes. Subcellular localization of LGP85 was determined using enzyme immunoassay. The lysosomes seem to be the major location. LGP85 in the lysosomes was divided into the membrane bound form (90%) and the soluble form (10%). Immunoelectron microscopy clearly confirmed that the localization of LGP85 is mainly confined to lysosomes.


Calcium-induced splitting of connectin filaments into beta-connectin and a 1,200-kDa subfragment.

When rabbit skeletal muscle myofibrils were treated with a solution containing 0.1 mM Ca2+ and 30 micrograms of leupeptin/ml, alpha-connectin, which forms very thin filaments in myofibrils, was split into beta-connectin and a 1,200-kDa subfragment. A part of beta-connectin located near the junction between beta-connectin and the subfragment seems to have an affinity for calcium ions and to be susceptible to the binding of large amounts of calcium ions. The calcium-binding site on beta-connectin is localized near the N2 line in the I band, and the subfragment is localized adjacent to the Z disk. It is possible that connectin filaments change their elasticity during the contraction-relaxation cycle of skeletal muscle at the physiological concentration of calcium ions. Because postmortem skeletal muscles lose their elasticity and become plastic in association with the calcium-specific splitting of connectin filaments, the splitting is considered to be a factor in meat tenderization during postrigor ageing.


Beta-oxidation of butyrate, the short-chain-length fatty acid, occurs in peroxisomes in the yeast Candida tropicalis.

When an n-alkane-utilizable yeast, Candida tropicalis pK233, was cultivated on butyrate, the fatty acid of shortest chain-length for beta-oxidation, as the sole source of carbon and energy, catalase and the enzymes of the fatty acid beta-oxidation system were inducibly synthesized at high levels. As in the alkane-grown cells, the proliferation of peroxisomes was harmonized with the induction of peroxisomal enzymes. The results of subcellular fractionation and immunoelectronmicroscopy indicated the localization of these enzymes in peroxisomes, not in mitochondria. It was suggested that only peroxisomes have a role in fatty acid beta-oxidation in the yeast cells, unlike in mammalian cells, in which cooperation between peroxisomes and mitochondria is essential.


Phosphorylation of histone H2A by protein kinase C and identification of the phosphorylation site.

In regenerating rat liver, nuclear protein histone H2A was shown to be phosphorylated on its amino-terminal serine residue [Sung et al. (1971) J. Biol. Chem. 246, 1358-1364], but the protein kinase which phosphorylates this residue has not been identified. To evaluate the possibility that protein kinase C can phosphorylate this residue, calf thymus histone H2A was 32P-labeled by incubation with [gamma-32P]ATP and highly purified protein kinase C from rat brain in the presence of calcium and phospholipid. About 1 mol of 32P was incorporated per mol of histone H2A and the Km and apparent Vmax of the reaction were calculated to be 2.1 microM and 0.35 mumol/min/mg, respectively. So histone H2A seemed to be a good substrate for protein kinase C. Further, the proteolytic phosphopeptides of 32P-labeled histone H2A were isolated by means of a series of column chromatographies and analyzed for their amino acid compositions. Comparison of the data with the known primary structure of histone H2A revealed their amino acid sequence as 1Ser-Gly-Arg. These data suggest that protein kinase C may be a candidate for the protein kinase which phosphorylates the amino-terminal serine residue of histone H2A during the regeneration of rat liver.


Studies on amino acid sequences of two isoforms of 17-kDa essential light chain of smooth muscle myosin from porcine aorta media.

Amino acid sequences were analyzed for two isoforms of myosin essential light chain, LC17a and LC17b [Hasegawa, Y., Ueno, H., Horie, K., & Morita, F. (1988) J. Biochem. 103, 15-18] from porcine aorta smooth muscle. Both LC17a and LC17b consisted of 150 amino acid residues and their N-terminal Cys residues were blocked by an acetyl group. The amino acid sequences of LC17a and LC17b were common from the N-terminal to Glu-141 and five amino acid substitutions were observed within the remaining C-terminal 9 residues. The amino acid sequences of LC17a and LC17b were identical to those deduced from the nucleotide sequences of bovine aortic cDNAs encoding the two isoforms [Lash, J. A., Helper, D.J., Klug, M., Nicolozakes, A.W., & Hathaway, D.R. (1990) Nucleic Acids Res. 18, 7176].


Mitosis-specific phosphorylation of the nuclear oncoproteins Myc and Myb.

The c-myc and c-myb proto-oncogenes encode phosphorylated nuclear DNA binding proteins that are likely to be involved in transcriptional regulation. Here we demonstrate that both Myc and Myb proteins are hyperphosphorylated during mitosis. In the case of Myb, hyperphosphorylation is accompanied by the appearance of three M phase-specific tryptic phosphopeptides. At least one of these phosphopeptides corresponds to a phosphopeptide generated after phosphorylation of Myb in vitro by p34cdc2 kinase. By contrast, the mitotic hyperphosphorylation of Myc does not correlate with the appearance of unique phosphopeptides, suggesting that M phase and interphase sites may be clustered within the same peptides. In addition Myc does not appear to be a target for p34cdc2 phosphorylation. The hyperphosphorylated forms of Myc and Myb from mitotic cells are functionally distinct from the corresponding interphase proteins in that the former have reduced ability to bind nonspecificially to double-stranded DNA cellulose. Furthermore, mitotic Myb binds poorly to oligodeoxynucleotides containing an Myb response element. We surmise that the decreased DNA binding capacity of hyperphosphorylated Myb and Myc during M phase may function to release these proteins from chromatin during chromosome condensation.


cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells.

A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase. Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level. By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase. Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm. During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes. The nuclear locale reappears during telophase. In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T. Hunter. 1991. J. Cell Biol. 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity.


The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre-Golgi compartment.

Evidence is accumulating that a distinct compartment(s) exists in the secretory pathway interposed between the rough ER (RER) and the Golgi stack. In this study we have defined a novel post-RER, pre-Golgi compartment where unassembled subunits of rubella virus (RV) E1 glycoprotein accumulate. When RV E1 is expressed in CHO cells in the absence of E2 glycoprotein, transport of E1 to the Golgi complex is arrested. The compartment in which E1 accumulates consists of a tubular network of smooth membranes which is in continuity with the RER but has distinctive properties from either the RER, Golgi, or previously characterized intermediate compartments. It lacks RER and Golgi membrane proteins and is not disrupted by agents which disrupt either the RER (thapsigargin, ionomycin) or Golgi (nocodazole and brefeldin A). However, luminal ER proteins bearing the KDEL signal have access to this compartment. Kinetically the site of E1 arrest lies distal to or at the site where palmitylation occurs and proximal to the low temperature 15 degrees C block. Taken together the findings suggest that the site of E1 arrest corresponds to, or is located close to the exit site from the ER. This compartment could be identified morphologically because it is highly amplified in cells overexpressing unassembled E1 subunits, but it may have its counterpart among the transitional elements of non-transfected cells. We conclude that the site of E1 arrest may represent a new compartment or a differentiated proximal moiety of the intermediate compartment.


In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties.

We have studied the effects of brefeldin A (BFA) on the tubular endosomes in AtT20 and HeLa cells (Tooze, J., and M. Hollinshead. 1991. J. Cell Biol. 115:635-653) by electron microscopy of cells labeled with three endocytic tracers, HRP, BSA-gold, and transferrin conjugated to HRP, and by immunofluorescence microscopy. For the latter we used antibodies specific for transferrin receptor, and, in the case of AtT20 cells, also antibodies specific for synaptophysin. In HeLa cells BFA at concentrations ranging from 1 micrograms to 10 micrograms/ml causes the dispersed patches of network of preexisting tubular early endosomes to be incorporated within 5 min into tubules approximately 50 nm in diameter but up to 40-50 microns long. These long, straight tubular endosomes are aligned along microtubules; they branch relatively infrequently to form an open network or reticulum extending from the cell periphery to the microtubule organizing center (MTOC). As the incubation with BFA is prolonged beyond 5 min, a steady state is reached in which many tubules are located in a dense network enclosing the centrioles, with branches extending in a more open network to the periphery. This effect of BFA, which is fully reversed within 15-30 min of washing out, is inhibited by pre-incubating the cells with sodium azide and 2-deoxy-D-glucose. In AtT20 cells BFA at 5 micrograms/ml or above causes the same sorts of changes, preexisting tubular endosomes are recruited into a more continuous endosomal network, and there is a massive accumulation of this network around the MTOC. Maintenance of the BFA-induced endosomal reticulum in both cell types is dependent upon the integrity of microtubules. In AtT20 cells BFA at 1 microgram/ml has no detectable effect on the early endosomal system but the Golgi stacks are converted to clusters of tubules and vesicles that remain in the region of the MTOC during prolonged incubations. Therefore, the Golgi apparatus in these cells is more sensitive to BFA than the early endosomes. The morphological evidence suggests that all the tubular early endosomes in BFA-treated HeLa and AtT20 cells are linked together in a single reticulum. Consistent with this, incubations as short as 1-3 min with 10 or 20 mg/ml HRP in the medium result in the entire endosomal reticulum in most of the BFA-treated cells being filled with HRP reaction product.(ABSTRACT TRUNCATED AT 400 WORDS)


The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals.

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.


In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.


2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells.

Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells is immobile as measured by FRAP. Previous studies suggest that the immobile fraction may be associated with the actin cytoskeleton (Pagliaro, L. and D. L. Taylor. 1988. J. Cell Biol. 107:981-991), and it has been proposed that the association of some glycolytic enzymes with the cytoskeleton could have functional significance, perhaps involving a fundamental relationship between glycolysis, cytoplasmic organization, and cell motility. We have tested the effect of a key glycolytic inhibitor and an actin cytoskeletal modulator on the mobility of aldolase in living cells directly, using fluorescent analog cytochemistry and FRAP. We report here that the competitive hexokinase inhibitor 2-deoxyglucose releases the bound fraction of aldolase in 3T3 cells within 10 min, and that this process is reversible upon washout of the inhibitor. A similar result is produced with the actin-binding agent, cytochalasin D. These results are consistent with models in which glycolytic enzymes are not exclusively diffusion-limited, soluble proteins, but may exist partially in the solid phase of cytoplasm. Such organization has significant implications for both the modulation of cytoplasmic structure and for cellular metabolism.


Tubulin protofilaments and kinesin-dependent motility.

Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility.