Normal Banding Pattern Research Articles

Chromosome Bandings and Recognition.

Chromosome banding can be defined as the lengthwise variation in staining properties along a chromosome stained with a dye. Chromosome banding became more practical in the early 1970s and is an essential technique used in karyotyping to identify human chromosomes for both clinical and research purposes. Most importantly, karyotyping is now considered a mandatory investigation of all newly diagnosed leukemias. Some banding methods, such as Giemsa (G)-, reverse (R)-, and centromere (C)-banding, still contribute greatly by being used as a routine procedure in clinical cytogenetic laboratory nowadays. Each chromosome has a unique sequence of bar code-like stripes, allowing the identification of individual homologues and the recognition of structural abnormalities through analyzing the disruption of the normal banding pattern at specific landmarks, regions, and bands as described in the ideogram. Since the quality of metaphases obtained from malignant cells is generally inferior to normal constitutional cells for karyotyping, a practical and accurate chromosome identification training guide is indispensable for a trainee or newly employed cytogenetic technologist in a cancer cytogenetic laboratory. The most common and currently used banding methods and chromosome recognition guide for distinguishable bands of each chromosome are described in detail in this chapter with an aim to facilitate quick and accurate karyotyping in cancer cells.

Excitatory afferent modulation of complex spike synchrony.

Inferior olivary neurons receive extensive glutamatergic and GABAergic innervation. Yet, because of the membrane properties of olivary neurons these neurotransmitters can produce only small changes in the firing rates of these cells. Moreover, olivary neurons can generate spontaneous spike activity in the absence of excitatory glutamatergic input. These facts suggest that glutamate and GABA have additional roles within the olivocerebellar system beyond simply modulating single cell firing probability. Indeed, one of the characteristics of the olivocerebellar system is its ability to generate synchronous complex spike activity across populations of Purkinje cells. The pattern of synchronous activity changes rapidly, and is thought to reflect the momentary distribution of effective electrotonic coupling between olivary neurons as shaped by afferent input to the inferior olive. However, it also possible that synchronous olivocerebellar activity is the result of synchrony inherent in the afferent activity itself. The issue of the origin of complex spike synchrony, and the role of glutamatergic olivary afferents in modulating its distribution were recently studied using multiple electrode recordings from Purkinje cells. The results of these studies, reviewed here, demonstrate that synchronous complex spike activity occurs in the absence of glutamatergic (and GABAergic) input to the inferior olive, and therefore indicate that synchronization of complex spike activity primarily results from the electrotonic coupling of olivary neurons, rather than from synchronization present within their afferents. Instead of triggering synchronous discharges directly, the results suggest that the function of tonic excitatory activity is to modulate the effective coupling of spike activity between olivary neurons. Blocking glutamate within the inferior olive causes an enhancement of the normal banding pattern of complex spike synchrony, with higher synchrony among parasagittally aligned Purkinje cells and less synchrony between non-aligned cells. This is in contrast to the more uniform synchrony distribution that follows block of GABAergic olivary afferents. Thus, GABA and glutamate play critical, and complementary, roles in determining the patterns of synchronous complex spike activity that are likely central to the functioning of the olivocerebellar system.

Organization of Olivocerebellar Activity in the Absence of Excitatory Glutamatergic Input

The olivocerebellar system has been proposed to function as a timing device for motor coordination in which inferior olivary neurons act as coupled oscillators that spontaneously generate rhythmic and synchronous activity. However, the inferior olive receives excitatory afferents, which can also drive the activity of these neurons. The extent to which the olivocerebellar system can intrinsically generate synchronous activity and olivary neurons act as neuronal oscillators has not been determined. To investigate this issue, multiple electrode recordings of complex spike (CS) activity were obtained from 236 crus 2a Purkinje cells in anesthetized rats. Intraolivary injections of the glutamate antagonists 6-cyano-7-nitroquinoxaline-2,3-dione or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium were made, and the resulting changes in CS activity were determined. Loss of evoked CS responses to motor cortex stimulation or perioral tactile stimulation was used to measure the efficacy of the block. Block of glutamatergic input decreased the average CS firing rate by approximately 50% but did not abolish spontaneous CS activity. The remaining CS activity was significantly more rhythmic than that in control. The patterns of synchrony were similar to those found in control conditions (i.e., synchronous CSs primarily occurred among Purkinje cells located within the same approximately 250-microm-wide rostrocaudally oriented cortical strip); however, this normal banding pattern was enhanced. These changes in CS activity were not observed with vehicle injections. The results suggest that excitatory afferent activity disrupts olivary oscillations and support the hypotheses that olivary neurons are capable of acting as neuronal oscillators and that synchronous CS activity results from electrotonic coupling of olivary neurons.

Additional Copies of the Proteolipid Protein Gene Causing Pelizaeus-Merzbacher Disease Arise by Separate Integration into the X Chromosome

The proteolipid protein gene (PLP) is normally present at chromosome Xq22. Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD). Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22. In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene. The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis. Another three affected males from the family had similar findings. In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome. In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22. A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern. The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders.

Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue.

All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage. In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. One compound, 1,9-dimethylmethylene blue (dimethyl-methylene blue), had superior screening results and was chosen for further characterization. White cell reduced RBC suspensions (30% hematocrit) were deliberately inoculated with extracellular virus or virus-infected VERO cells, incubated with 4 microM dimethyl-methylene blue and illuminated with cool-white fluorescent light. Control and treated samples were titered for virus inactivation. In parallel studies, RBC suspensions were exposed to dimethylmethylene blue and light under identical conditions and assayed for in vitro RBC storage properties. Phototreatment of RBC suspensions inactivated > 4.4 log10 of extracellular vesicular stomatitis virus (VSV), > 3.0 log10 of intracellular VSV, > 5.0 log10 of extracellular pseudorabies virus (PRV), > 4.8 log10 of intracellular PRV, > 4.7 log10 of extra-cellular bovine virus diarrhea virus, 5.8 log10 of bacterio-phage phi 6 and > 7 log10 of bacteriophage R17. Encephalo-myocarditis virus, a nonenveloped picornavirus, was resistant to photoinactivation. Virucidal conditions resulted in no detectable IgG binding in 11 of 13 samples, unchanged RBC morphology, normal banding patterns of RBC membrane proteins on SDS PAGE, and unaltered characteristics of 12 of 13 RBC antigens during storage as measured by antibody titrations. In addition, minimal changes were observed in RBC osmotic fragility, lysis, potassium efflux, ATP and 2,3-DPG levels, and the strength of one RBC antigen during storage of phototreated samples compared with controls. Dimethylmethylene blue photo-treatment can inactivate several intracellular and extracellular model viruses under conditions which minimally alter RBC properties during 42 days storage at 1-6 degrees C.

Rostral cerebellar malformation (rcm/rcm): a murine mutant to study regionalization of the cerebellum.

A recently described recessive mouse mutant, rostral cerebellar malformation (rcm/rcm), demonstrates a swaying gait at approximately 12 days of age (Lane et al. [1992] J. Hered. 83:315-318). The mutant cerebellar (Cb) phenotype consists of cerebellar tissue that extends rostrally, beyond the usual distinct anterior cerebellar boundary, into the midbrain (Lane et al. [1992] J. Hered. 83:315-318; Ackerman et al. [1997] Nature 386:838-842). Interestingly, the cerebellar ectopia occurs in the absence of any significant alterations in the distribution of nuclear groups within the brainstem. The ectopic Cb tissue is 1) adherent to the posterior and lateral aspects of the inferior colliculus and to the lateral aspect of the rostral brainstem and 2) contains acellular regions within the inner granular layer (igl) and ectopic, calbindin-immunoreactive Purkinje cells (PCs) deep to the igl. Within the Cb proper, PC organization, as revealed by zebrin II immunoreactivity, is generally normal. In the ectopic Cb tissue PCs also exhibit a banded zebrin distribution. Analysis of the spinocerebellar projection in the mutant suggests a lobular distribution similar to that seen in the normal mouse. Within the anterior region, however, the normal parasagittal banding pattern is somewhat obscured. Spinocerebellar innervation of the ectopic Cb tissue exists, but it is almost exclusively to the region adjacent to the caudal inferior colliculus. In conjunction with the recent finding that the mutation appears to affect a UNC-5-like receptor protein for netrin-1, a molecule that may be involved in axonal guidance and cell migration (Ackerman et al. [1997] Nature 386:838-842), our results suggest that this mutant is an important model for the analysis of cerebellar development and regionalization.

The effects of nucleoside analogs on telomerase and telomeres in Tetrahymena.

The ribonucleoprotein enzyme telomerase is a specialized type of cellular reverse transcriptase which synthesizes one strand of telomeric DNA, using as the template a sequence in the RNA moiety of telomerase. We analyzed the effects of various nucleoside analogs, known to be chain-terminating inhibitors of retroviral reverse transcriptases, on Tetrahymena thermophila telomerase activity in vitro. We also analyzed the effects of such analogs on telomere length and maintenance in vivo, and on vegetative growth and mating of Tetrahymena cells. Arabinofuranyl-guanosine triphosphate (Ara-GTP) and ddGTP both efficiently inhibited telomerase activity in vitro, while azidothymidine triphosphate (AZT-TP), dideoxyinosine triphosphate (ddITP) or ddTTP were less efficient inhibitors. All of these nucleoside triphosphate analogs, however, produced analog-specific alterations of the normal banding patterns seen upon gel electrophoresis of the synthesis products of telomerase, suggesting that their chain terminating and/or competitive actions differ at different positions along the RNA template. The analogs AZT, 3'-deoxy-2',3'-didehydrothymidine (d4T) and Ara-G in nucleoside form caused consistent and rapid telomere shortening in vegetatively growing Tetrahymena. In contrast, ddG or ddI had no effect on telomere length or cell growth rates. AZT caused growth rates and viability to decrease in a fraction of cells, while Ara-G had no such effects even after several weeks in culture. Neither AZT, Ara-G, acycloguanosine (Acyclo-G), ddG nor ddI had any detectable effect on cell mating, as assayed by quantitation of the efficiency of formation of progeny from mated cells. However, AZT decreased the efficiency of programmed de novo telomere addition during macronuclear development in mating cells.

Cytogenetic and molecular aspects of position-effect variegation in Drosophila melanogaster. V. Heterochromatin-associated protein HP1 appears in euchromatic chromosomal regions that are inactivated as a result of position-effect variegation.

Position-effect variegation in Drosophila melanogaster is accompanied by compaction of the corresponding chromosomal regions. The compaction can be continuous, so that bands and interbands located distal to the eu-heterochromatic junction fuse into one dense block, or discontinuous, when two or more zones of compaction are separated by morphologically and functionally normal regions. In this work it was found that in both continuous and discontinuous compaction the blocks of dense material contain the immunochemically detectable protein HP1, which has previously been characterized as specific for heterochromatin. The regions undergoing compaction do not contain HP1 when they have a normal banding pattern. Thus, it may be proposed that HP1 is one of the factors involved in compaction. If two different or two identical rearrangements are combined in the same nucleus, they variegate independently. The frequency of compaction of the two rearrangements in the same nucleus corresponds to the product of the frequencies of the compact state of the individual elements. The extent of compaction (i.e. the number of bands involved in heterochromatization) of each rearrangement does not depend on the compaction pattern of the other rearranged element.

The Degradation of Type II Collagen in Rheumatoid Arthritis: An Immunoelectron Microscopic Study

Rabbit antibodies were prepared that react only with denatured type II collagen alpha-chains and cleavage products. The epitopes that these antibodies recognize reside in cyanogen bromide peptides CB8 and CB11. The antibodies do not react with triple helical collagen nor with any other collagen or protein present in hyaline cartilage (Dodge and Poole, J. Clin. Invest. 83:647-661, 1989). These antibodies can therefore be used to detect denatured type II collagen produced, for example, by enzymatic cleavage. In this study they were used to determine, at the ultrastructural level, using immunogold staining, type II collagen fibril cleavage in articular cartilages remote from synovium and pannus of patients with rheumatoid arthritis. Comparisons were made with site- and age-matched healthy articular cartilages. Antibody binding was detected in the extracellular matrix, at the articular surface and in the deep zone, usually on visibly damaged collagen fibrils which exhibited a loss of the normal banding pattern of staining produced by lead citrate and uranyl acetate: binding was also observed in disrupted fibrils, sometimes at their ends. Binding was commonly associated with amorphous-looking material (and occasionally unstained sites) in the extracellular matrix which, because of the specificity of the antibody, can be identified as containing denatured or fragmented type II collagen, stained (and unstained) with heavy metals. In both rheumatoid and healthy articular cartilages, there was no antibody binding to intact well stained fibrils which exhibited a regular banding pattern. Little or no staining was detected at the ultrastructural level in normal cartilages.(ABSTRACT TRUNCATED AT 250 WORDS)

Some Effects of Gamma Irradiation on Patellar Tendon Allografts

The purpose of this study was to investigate possible causes for altered mechanical properties of patellar tendon allografts, used for reconstructive surgery, after sterilization with 2.0 Mrads gamma irradiation. The tissues were analyzed using a Hydrothermal Isometric Tension (HIT) device in conjunction with biochemical assays of soluble collagen and electron microscopy to understand the action of this processing. Gamma irradiation was found to significantly lower the shrinkage temperature and tended to increase the rate of isometric tension development. While no significant alteration in the acetic acid solubility was noted, the processing doubled the solubility of collagen to pepsin. This could mean that the predominant effect of the gamma irradiation at this dose level is polypeptide chain scission. The stiffening of the HIT curve may suggest some minor cross linking also occurred. The damaging effect of gamma irradiation, however, was not significant enough to cause any major disruption of the normal banding pattern in collagen. These data support earlier biomechanical data showing that using current processing methods, 2.0 Mrads of gamma irradiation sterilization significantly decreases the tensile strength and modulus of human patellar tendon graft tissues.

Immunochemical analysis of uridine diphosphate-glucuronosyltransferase in four patients with the Crigler-Najjar syndrome type I.

The functional heterogeneity of uridine diphosphate-glucuronosyltransferase (UDPGT) and its deficiency in human liver were investigated. The monoclonal antibody (MAb) WP1, which inhibits bilirubin and phenol-glucuronidating activity, was used to immunopurify UDPGTs from human liver. Purified UDPGTs were injected into mice to obtain new MAbs. Immunoblotting of microsomes with MAb HEB7 revealed at least three polypeptides in liver (56, 54, and 53 kD) and one in kidney (54 kD). In liver microsomes from four patients (A, B, C, and D) with Crigler-Najjar syndrome type I (CN type I), UDPGT activity towards bilirubin was undetectable (A, B, C, and D) and activity towards phenolic compounds and 5-hydroxytryptamine either reduced (A and B) or normal (C and D). UDPGT activity toward steroids was normal. Immunoblot studies revealed that the monoclonal antibody WP1 recognized two polypeptides (56 and 54 kD) in liver microsomes from patient A and none in patient B. With HEB7 no immunoreactive polypeptides were seen in these two patients. Patient C showed a normal banding pattern and in patient D only the 53-kD band showed decreased intensity. These findings suggest considerable heterogeneity with regard to the expression of UDPGT isoenzymes among CN type I patients.

Retention of lens specificity in long-term cultures of diploid rabbit lens epithelial cells

Rabbit lens epithelial cells from newborn animals exhibited limited growth when cultured under standard conditions. Cell lines were generated when explants from individual lenses were cultured in medium supplemented with conditioned medium or untreated rabbit serum. All lines exhibited a stable epithelial morphology. One line, N/ N1003A. was examined extensively with respect to its growth, ploidy, and maintenance of lens-specific functions. Cells at population-doubling level (pdl) 120 exhibited a normal chromosomal banding pattern, were diploid, were non-tumorigenic in vivo, did not grow in suspension culture, and did not exhibit sustained growth in medium supplemented with low concentrations of serum. The shape of the growth curves and the final density for cells at pdl 24 and 181 exposed to various concentrations of serum were identical. The cells showed no diminution in growth as a function of in vitro age. The cells retained lens-specific functions. Proteins were isolated from cells at pdl 40 and 170, and were separated on polyacrylamide gels. Western immunoblot analysis using antiserum to α-crystallin, a tissue-specific protein found in lens epithelial cells in vivo, indicated the presence of α-A- and α-B-crystallin polypeptides. The cells also contained the transcription factors required for activating the murine α-A-crystallin gene promoter. which is known to function with precise tissue specificity. When an expression vector including the bacterial chloramphenicol acetyltransferase (CAT) gene controlled by the α-A-crystallin gene promoter was introduced into the lens epithelial cells, the CAT gene was expressed. This did not occur when this construct was introduced into cultured rabbit kidney epithelial cells. Cultured lens epithelial cells provide a stable system for investigating the factors that regulate growth, differentiation, and crystallin gene expression in lens epithelial cells.

Effect of colchicine on the pattern of ameloblast modulation visualized by the glyoxal bis (2-hydroxyanil) staining and calcein labeling in rat incisors.

The present study was undertaken to investigate the effect of the microtubules inhibitor, colchicine, on the cyclic modulation pattern of the maturation ameloblasts in rat incisors using the glyoxal bis (2-hydroxyanil) (GBHA) staining method and fluorescent labeling with calcein. Rats were killed at intervals of 2, 4, 8, 12, 24 and 48 hours after the subcutaneous (s. c.) injection of colchicine at a dose of 1.3mg/kg, and at 8 hours after the injection (s. c.) of lumicolchicine or physiological saline (control). All rats were injected intravenously with calcein at a dose of 15mg/kg 1 hour before killing. Several red bands stained with GBHA and several yellow-green fluorescent bands labeled with calcein were observed on the maturing enamel surface. After the colchicine injection, these bands were disarranged and areas that stained with GBHA or labeled with calcein increased to about 50% of the maturing enamel surface at 8 and 24 hours. By 48 hours after the injection, these areas were reduced and showed a normal banding pattern again. Lumicolchicine, an isomer of colchicine which does not disrupt the microtubules, did not affect the pattern of GBHA staining and calcein labeling bands. These observations suggest that the cytoplasmic microtubular function of maturation ameloblast is important for the cyclic modulation in rat incisor.

Retention of lens specificity in long-term cultures of diploid rabbit lens epithelial cells

Rabbit lens epithelial cells from newborn animals exhibited limited growth when cultured under standard conditions. Cell lines were generated when explants from individual lenses were cultured in medium supplemented with conditioned medium or untreated rabbit serum. AH lines exhibited a stable epithelial morphology. One line, N/ N1003A, was examined extensively with respect to its growth, ploidy, and maintenance of lens-specific functions. Cells at population-doubling level (pdl) 120 exhibited a normal chromosomal banding pattern, were diploid, were nontumorigenic in vivo, did not grow in suspension culture, and did not exhibit sustained growth in medium supplemented with low concentrations of serum. The shape of the growth curves and the final density for cells at pdl 24 and 181 exposed to various concentrations of serum were identical. The cells showed no diminution in growth as a function of in vitro age. The cells retained lens-specific functions. Proteins were isolated from cells at pdl 40 and 170, and were separated on polyacrylamide gels. Western immunoblot analysis using antiserum to α-crystallin, a tissuespecific protein found in lens epithelial cells in vivo, indicated the presence of α-A- and α-B-crystallin polypeptides. The cells also contained the transcription factors required for activating the murine α-A-crystallin gene promoter, which is known to function with precise tissue specificity. When an expression vector including the bacterial chloramphenicol acetyltransferase (CAT) gene controlled by the α- A-crystallin gene promoter was introduced into the lens epithelial cells, the CAT gene was expressed. This did not occur when this construct was introduced into cultured rabbit kidney epithelial cells. Cultured lens epithelial cells provide a stable system for investigating the factors that regulate growth, differentiation, and crystallin gene expression in lens epithelial cells.

Clonal separation of mature melanocytes from premelanocytes in a diploid human cell strain: spontaneous and induced pigmentation of premelanocytes.

Strains of pigmented melanocytes can be derived reproducibly from normal human skin. Published procedures have been modified here to yield a strain, 'Nohm-1', comprising many unpigmented cells as well as cells with various degrees of pigmentation observable by light microscopy. The unpigmented cells contain early melanosomes (pigment organelles) and the specific enzyme tyrosinase. They are on average smaller and less dendritic than the pigmented cells. Nohm-1 cells show normal chromosomal banding patterns and normal proliferative behaviour, including senescence. They form no tumours in immunodeficient (nude) mice. Nohm-1 cells have been cloned and yield two distinct types of colony, depending on the progenitor cell. Well-pigmented melanocytes engender pure colonies of pigmented cells, but cells with little or no pigment can produce both unpigmented and pigmented progeny. Thus there is a separate cell type, or premelanocyte, which can differentiate spontaneously and stably in culture; this cell type includes both unpigmented and faintly pigmented cells. Usefully, most premelanocytes are viable after frozen storage, unlike well-pigmented melanocytes. Some components of the culture medium affect the proportion of pigmented cells in Nohm-1 cultures, and hence probably the ratio of mature melanocytes to premelanocytes. Rapid pigmentation can be induced artificially and simply, by using a medium with increased extracellular pH and tyrosine concentration.

Correlation between tumorigenicity and banding pattern of chromosome 15 in murine T-cell leukemia cells and hybrids of normal and malignant cells.

The most common chromosomal aberration in murine T-cell lymphomas is trisomy of chromosome 15. It has now been shown that the chromosomal region 15E exhibits a variant early replication banding pattern after 5-bromo-deoxyuridine labeling during part of the preceding S-phase. This variation is restricted to T-cell tumors. Plasmacytomas bearing the specific translocation t(12;15) show a normal early replication banding pattern of chromosome 15. In T-cell tumors all three chromosomes 15 of one cell are of the same variant banding type. In hybrids between tumor and nontumor cells, the number of cells expressing the variant early replication banding pattern is related to the degree of malignancy. Chromosomes 15 in one cell never expressed the variant and the normal banding pattern simultaneously. All five to six chromosomes 15 from one hybrid cell are of the same banding type irrespective of their parental origin. With respect to the type of early replication banding pattern, there is complete reversibility; tumor-parent-derived chromosomes 15 change to normal, and normal-parent-derived chromosomes 15 change to tumor.

Eccentric exercise-induced injury to rat skeletal muscle

These experiments were designed to study skeletal muscle pathology resulting from eccentric-biased exercise in rats. The effects on the muscles of running on a treadmill on a 0 degrees incline (similar amounts of concentric and eccentric contractions), down a 16 degrees incline (primarily eccentric contractions), and up a 16 degrees incline (primarily concentric contractions) at 16 m . min-1 for 90 min were assessed by following postexercise changes in 1) plasma creatine kinase and lactate dehydrogenase activities, 2) glucose-6-phosphate dehydrogenase (G-6-PDase) activity (bio- and histochemically) in the physiological extensor muscles, and 3) histological appearance of the muscles. The data indicate the following. 1) Whereas all exercise protocols resulted in elevations of plasma enzymes immediately after running, only eccentric exercise caused late phase elevations 1.5-2 days postexercise. 2) Significant increases in muscle G-6-PDase activity, which were always associated with accumulations of mononuclear cells, always occurred within some muscles of each extensor group 1-3 days following downhill and uphill running and did not occur following level running; the increases in activity were usually of lower magnitude in the muscles of uphill runners than in those of downhill runners; the deeply located, predominantly slow-twitch muscles were most affected by both down- and uphill running. 3) Muscle histology demonstrated localized disruption of normal banding patterns of some fibers immediately after exercise and accumulations of macrophages in the interstitium and in some (less than 5%) muscle fibers by 24 h postexercise in the deep slow muscles of the antigravity groups. Although the data generally indicated that eccentric exercise causes greater injury to the muscles, questions remain.

Analysis of erythrocyte membrane proteins in a variant case of congenital dyserythropoietic anaemia (CDA) type II

We have had the opportunity to carry out biochemical studies on the erythrocytes of a patient with an unusual variant of CDA II. As previously reported, differences from typical cases include the presence of large numbers of normoblasts in the peripheral blood, a high percentage of erythrocytes with reduplication of the membrane, lack of the HEMPAS antigen, rare Kell serology (Kp b -negativity) and clinically, severe tophaceous gout. Erythrocytes from normal volunteers and the patient were lysed and washed ghosts were prepared and analysed on SDS-polyacrylamide gels. Alterations in the normal banding pattern of erythrocyte membrane components were seen in the patient, notably the absence of a component in the B region (MW 100000) and a marked increase in the amount of a C region component (MW 50000). These findings are essentially similar to those reported for more typical CDA II cases. It is not clear whether the altered pattern reflects directly the composition of the erythrocyte membrane in vivo in the disease, or whether the changes represent a greater susceptibility of the membrane to proteolytic enzymes in CDA II. Further experiments are in progress.

VARIATION IN THE ABDOMINAL STERNAL BANDING OF CULEX ANNULIROSTRIS SKUSE (DIPTERA: CULICIDAE)

SUMMARYMosquitoes identified as Culex sp. nr annulirostris, or termed atypical in Annual Reports of the Queensland Institute of Medical Research, and with uninterrupted apical dark banding on abdominal sternites V‐VII, are variants of Cx annulirostris. Atypical progeny were reared from females of normal banding pattern and vice versa. The atypical pattern occurred 5‐12 times more often in males than females. An average of 2% of female Cx annulirostris sampled from 23 localities in Queensland and two in the Northern Territory were of the atypical pattern, which occurred more in the north than in arid inland centres.

Long-term culture of Chinese hamster Kupffer cell lines isolated by a primary cloning step

The purpose of this study was to isolate many cell lines of defined histotypic origin from the one animal. Once isolated, such material could be used for a study of phenotypic variation of cells in culture. Non-parenchymal liver cells were isolated from Chinese hamsters by enzymatic digestion and centrifugation. The resulting cell suspensions were cultured and cell lines isolated by a primary cloning step. Those clones which exhibited peroxidase (EC 1.11.1.7), microsomal haem oxygenase (EC 1.14.99.3) and phagocytic activities were considered to be derived from Kupffer cells and retained for further culture. The Kupffer cell lines could be cultured for extended periods and some survived 97 population doublings. The Kupffer cell functions of peroxidase, microsomal haem oxygenase and phagocytic activity persisted until at least 55 population doublings in culture. Thereafter there tended to be an ordered pattern of loss of these functions. Loss of phagocytic activity always preceded the loss of microsomal haem oxygenase, whilst peroxidase activity persisted throughout the study period. Karyotype analysis revealed that the cell lines were diploid for at least 50 population doublings and that the chromosomes retained a normal banding pattern. The fact that 130 Kupffer cell lines were isolated by a primary cloning step from three animals suggests that this system could be of value in studies of both cultured Kupffer cells and phenotypic variation of identified, differentiated cells in culture.