The ultrastructure of the chromosome periphery in human cell lines. An in situ study using cryomethods in electron microscopy.

We studied the chromosome periphery in human HeLa and TG cells using cryomethods in electron microscopy. A contrasted layer of peripheral chromosomal material (PCM) was visible in cryo-ultrathin sections of mitotic cells. This PCM was composed of closely packed fibrils associated with granules. The PCM did not cover the entire chromosome surface but was found around most of the chromosomes and even between two chromatids. The organization of the PCM was not affected by colchicine treatment of mitotic cells. In cells prepared by quick-freezing, the PCM appeared to be a fibrous material at the chromosome periphery, and was also associated with granules that resembled inter-chromatin granules in size and shape. At higher magnification, direct contacts between the chromosomes and the fibrils of the PCM were observed. The cryotechniques used are known to preserve the native organization of cells. Therefore, the architecture of the perichromosomal region analysed presumably corresponds to that in vivo during mitosis. These observations show that in HeLa and TG cells, a particular structure present at the chromosome periphery in the form of PCM is persistent and ubiquitous. In addition, we showed by immunolabelling that the PCM is the specific site of accumulation of nucleolar antigens during mitosis. These two results, i.e. the identification of specific morphological structures and the compartmentation of proteins, indicate that this layer is a specific region of mitotic cells.

Identification and characterization of a new set of nucleolar ribonucleoproteins which line the chromosomes during mitosis.

We investigated the perichromosomal architecture established during mitosis. Entry into mitosis brings about a dramatic reorganization of both nuclear and cytoplasmic structures in preparation for cell division. While the nuclear envelope breaks down, nuclear proteins are redistributed during chromosome condensation. Some of these proteins are found around the chromosomes, but little is known concerning their nature and function. Ten autoimmune sera were used to study the microenvironment of chromosomes and, in particular, the chromosome periphery. They were selected for their anti-nucleolar specificity and were found to recognize three nucleolar proteins that coat the chromosomes during mitosis. The distribution of these antigens was followed through the cell cycle by confocal laser scanning microscopy. The antigens dispersed very early during prophase and simultaneously with the chromosome condensation suggesting a correlation between these two processes. The antigens have apparent molecular weights of 53, 66, and 103 kDa on SDS-PAGE migration. Elution of the antibodies and immunopurification showed that they are RNA-associated proteins. The coimmunoprecipitating RNA moiety involved in these RNPs appeared to be U3, but the antigens are not related to the fibrillarin family. Therefore, small nucleolar RNPs follow the same distribution during mitosis as that described for small nuclear RNPs. Possible functions for these antigens are discussed.

A 116,000 Mr nucleolar antigen specific for the dense fibrillar component of the nucleoli.

In ATT, a human autoimmune serum, we found anti-nucleolar antibodies that recognized nucleolar antigens confined to a single nucleolar compartment, the dense fibrillar component (DFC). We localized these antigens by immunoelectron microscopy in DFC of HeLa cell nucleoli both on Lowicryl sections and cryoultrathin sections without embedding. The antigens were solubilized by incubation with 2M NaCl but not by RNase or DNase treatment. The ATT serum crossreacted with rat liver nucleoli and PtK1 cell nucleoli in which immunofluorescence labelling displayed a clumpy pattern. During mitosis, the antigens dispersed in the cytoplasm until late telophase, when they gathered in the prenucleolar bodies. In human peripheral lymphocytes, or HeLa cells treated with actinomycin D, the antigens were still present but the fluorescence intensity decreased. By immunoblotting using human nuclear extracts, the ATT serum bound to a 116,000 Mr protein at dilutions up to 1:2000. The reactivity of this band diminished with actinomycin D-treated nuclear extracts. Two minor bands were also observed at 97 and 70K (K = 10(3) Mr). Immunopurification by competition or elution demonstrated that the 116K antigens were at the origin of the nucleolar labelling. This DFC marker appeared to be different from the NOR-silver-stained proteins, which in our preparations exhibited apparent molecular weights of 105, 80 and 38-40K. In addition, these 116K antigens did not exhibit the characteristics described for DNA topoisomerase I, fibrillarin or nucleolin. We propose the 116K antigen as a new marker of the DFC of the nucleoli.

Three-dimensional organization of micronuclei induced by colchicine in PtK1 cells.

In PtK1 cells micronucleated by colchicine, we previously demonstrated that some micronuclei contain a single chromosome. Here, we investigated interphase chromosome organization in micronucleated PtK1 cells using conventional electron microscopy and three-dimensional computer reconstruction. The distribution of micronuclei was not always polarized, but in some cells they formed a ring. When this occurred, centrioles and Golgi apparatus were located inside the ring. On freeze-fracture replicas, we observed that nuclear pore distribution among the micronuclei was heterogeneous, and on thin sections some micronuclei displayed an incomplete nuclear envelope, with gaps in the double membrane and areas without lamina or condensed chromatin. By autoradiography, we showed that the fibrillar dots were not sites of active transcription. We applied three-dimensional reconstruction to one micronucleated cell containing 22 micronuclei whose size indicated that each micronucleus probably contained one chromosome. In this cell we demonstrated that only the smallest micronuclei had an incomplete nuclear envelope. The presence in micronuclei of either nucleoli or fibrillar dots was found to be mutually exclusive. These dots might constitute stores of nucleolar proteins which migrate into micronuclei possessing no ribosomal genes. In NOR-bearing micronuclei, the structural organization was similar to that of diploid nuclei: the nucleoli were attached to the nuclear membrane and a nucleolar canal was seen, even in single-chromosome spherical micronuclei. Taken together, these findings indicate that in the diploid nuclei of PtK1 cells, the three-dimensional organization of the nucleolar domain seems to be directly controlled by the X-chromosome.

Goniotomy with sodium hyaluronate.

A modification of the classical goniotomy technique is described. After complete evacuation of the anterior and posterior chambers, sodium hyaluronate is placed in the anterior chamber and on the cornea. Putting hyaluronate in both locations prevents the formation of air bubbles under the goniotomy lens, provides the same index of refraction on both sides of the cornea, prevents accidental loss of the anterior chamber, and allows maximal depth of the anterior chamber. Diminished intraocular bleeding and lower risk of injury to the corneal endothelium, iris, and lens may be additional advantages of the procedure. Two cases are presented in which five goniotomies were performed on four eyes using this technique. Both patients had one eye controlled with a single goniotomy. The fellow eye of each patient required multiple procedures. Glaucoma was controlled successfully with no medication in all four eyes. No significant complications occurred.

Monocanalicular lacrimonasal intubation.

A new technique to obtain patency of the lacrimal drainage system was used in three children with bilateral congenital obstruction of the lacrimonasal duct (six cases) and upper canalicular abnormalities (five cases). The upper canalicular abnormalities prevented bicanaliculonasal intubation.A silicone tube was passed through the lower canaliculus, traversing the lacrimal drainage system and into the nasal fossa. The proximal end of the tube was placed subconjunctival in the inferior fornix and secured. The distal end of the tube was passed through the subcartilaginous nasal septum to the contralateral nasal fossa and tied to a tube similarly passed through the opposite lacrimal system.The procedure was successful in all six cases, with all tubes remaining in place the desired length of time. Postoperative identification and removal of the tubes offered no difficulties and was followed by permanent patency of the lacrimal drainage system in all six cases.

Effectiveness of botulinum toxin therapy for essential blepharospasm.

The effectiveness of Botulinum toxin (Oculinum) therapy in 76 patients with the diagnosis of essential blepharospasm was analyzed. Botulinum offers relief to almost all patients suffering from essential blepharospasm, however, this relief is usually temporary. The response time for repeated treatments tended to be longer than the first treatment. Patients with mild blepharospasm responded significantly longer to Botulinum injection, than those with severe spasms. The response to Botulinum was not significantly different in patients with Meige syndrome than in patients with only essential blepharospasm. Patients previously treated surgically for essential blepharospasm did not respond differently than those patients with no previous surgical therapy. The authors believe that Botulinum toxin injection is an effective, although temporary, mode of therapy for the signs and symptoms of this focal dystonia. The authors recognize that there may be psychologic factors affecting the response.

Comparative evaluation of current ophthalmodynamometers.

Seventy-five patients under 40 years of age with no history of ocular, cardiovascular, or cerebrovascular disease were examined with four commonly used ophthalmodynamometers. There was a significant difference in the absolute values measured with each instrument and the ophthalmic to brachial artery pressure ratio. The standard deviations were comparable between 8.1 and 10.2%. The absolute value of the average percent difference between the right and the left eye ranged from 3.6% with the Galin unit to 6.7% with the Sisler unit. Limits of +/- 15% for ophthalmic artery to brachial artery pressure ratio and 10% for right to left pressure difference should be used for 95% confidence levels. There was a small but definite correlation between the ocular coefficient of rigidity and pressure differences between direct and indirect instruments.

Growth of cytomegalovirus at supra-optimal temperatures.

An isolate (Mira) of cytomegalovirus is shown to replicate in human embryonic lung fibroblasts at supra-optimal temperature (40 degrees C). The ability of the Mira isolate to grow at 40 degrees C decreased as a function of age of cells in which the virus was grown. The unusual morphology of the lesions in late passage cells infected and maintained at 40 degrees C is illustrated.

Comparative study of the surgical management of congenital esotropia of 50 prism diopter or less.

Fort-nine patients with congenital esotropia of 50 prism diopters or less were selected at random. Twenty-five had bimedial recessions performed initially followed by bilateral rectus resections when needed. Twenty-four had recession/resections performed initially and when needed, were followed by the same procedure on the fellow eye. The average corrections obtained after first procedure and after second procedure were compared and analyzed. The results showed the initial procedures of both groups to be equally effective. The results obtained after the second procedure in the recession/resection group were significantly better. Therefore, it would appear that for the patient we have described, the most effective surgical approach is ultimately the surgical regimen of recession/resection followed by the same procedure on the fellow eye.

Cytomegalovirus infection of human lung epithelial cells in vitro.

Human lung epithelial cells were productively infected with human cytomegalovirus in vitro. Infectious virus was released up to 8 weeks postinfection. The cells retained their morphological characteristics throughout the period of observation, while simultaneously bearing all the features typical of cytomegalovirus infection.