Characteristic Banding Patterns Research Articles

Screening for hydroxyethyl starch (HES) doping in sport

The artificial colloid hydroxyethyl starch (HES) is among the most frequently used plasma volume expanders in the medical field. However, in 1998, its misuse by the athletic community was officially reported and since 2000, HES is prohibited by the International Olympic Committee (IOC). Therefore, several methods enabling the detection of HES in urine were developed, most based on gas chromatography–mass spectrometry (GC–MS). In the present work, a simple and low-cost screening method, intended to reduce the number of samples to be analysed by GC–MS, was developed. The method is based on the acid hydrolysis of HES and detection of the resulting glucose and hydroxyethyl glucose derivatives by Benedict's reaction (reduction of copper sulfate to brick-red cuprous oxide by glucose and/or derivatives). Samples considered suspect were submitted to GC–MS analysis for identification of HES. The method was successfully applied for screening of HES in 2627 urine samples from 1346 Brazilian soccer players and 1281 athletes from the Pan-American Games (Rio de Janeiro, 2007); 71 (2.7%) samples, considered suspect, were submitted to GC–MS, but no positive results were found. Moreover, a thin layer chromatography (TLC) method was adapted for visualisation of the characteristic band pattern of HES hydrolysis products. The results indicate that the methods are efficient and useful for the screening of HES in urine.

Human Lymphocyte Culture and Chromosome Analysis

INTRODUCTIONPhytohaemagglutinin (PHA), a lectin derived from the red kidney bean, is a powerful mitogen for human T-cells. When PHA is added in vitro to whole blood, mitotic cells can be found after 48 h, with a peak mitotic index at ~64-72 h. The convenience of peripheral blood as a source of human cells, the abundance of mitotic cells, and the simplicity of the cell culture technique make this the most convenient approach to study human chromosomes for both clinical and research purposes. This method of chromosome preparation provides metaphase cells that can be stained by a variety of methods or used for fluorescence in situ hybridization (FISH). The most common chromosome staining techniques involve exposing fixed preparations to a protease (e.g., trypsin), followed by an appropriate semipermanent stain. The characteristic banding patterns obtained reflect both structural and functional differences in different parts of the chromosomes. The staining procedure described here provides a Giemsa banding pattern using trypsin with Wright stain (i.e., GTW banding). This procedure is reliable and, with only minor modifications, suitable for preparing chromosomes from a variety of human tissues.

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise ( Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise ( Gopherus agassizii), and anti- M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.

Differentiation of Staphylococci from Iberian dry fermented sausages by protein fingerprinting

The Staphylococci populations in different types of Iberian dry fermented sausages from central-west Spain were identified. A simple electrophoretic method of whole-cell proteins and extracellular protein profiling was evaluated for speed of identification. This study was correlated with a 16S rRNA gene sequence analysis and biochemical identification by API Staph. A total of 81 isolates were identified by SDS–PAGE of the whole-cell proteins. These showed stable profiles in the range 99–14 kDa that were clearly different for the different species, and were grouped into clusters together with the profiles of the eight reference strains. SDS–PAGE of the extracellular protein extracts provided additional characteristic banding patterns for the characterization of the Staphylococcus species present. The whole-cell SDS–PAGE showed that the predominant species was Staphylococcus saprophyticus (61.7%) followed by Staphylococcus aureus (19.7%). The identifications were confirmed by the 16S rRNA gene sequencing and by a BLAST search of the GenBank database. However, the API Staph biochemical identifications were frequently erroneous at the species level. In sum, SDS–PAGE analysis showed itself to be rapid and accurate in identifying the most commonly encountered Staphylococcus isolates in dry fermented sausages.

Molecular characterization of the diversity of Clostridium chauvoei isolates collected from two bovine slaughterhouses: Analysis of cross-contamination

Clostridium chauvoei is the etiologic agent of blackleg, a high mortality rate disease affecting mainly cattle and sheep. Carcasses of animals affected by the disease are the chief source of soil infection and considered as an ever-present threat to livestock health. A study was undertaken to examine the cross-contamination of C. chauvoei in two different bovine slaughterhouses using restriction endonuclease analysis (REA) and protein analysis. Samples from various sites of two different bovine slaughterhouses were screened and 34 isolates were identified by conventional techniques and 16S rRNA gene ( rrs) sequencing. C. chauvoei were isolated from carcass, soil, and sewage from slaughterhouses examined. The isolates were differentiated using REA and whole-cell and excretory protein pattern analysis combined with numerical analysis and cluster formation. The α and β toxins produced by the strains were characterized. Our preliminary results suggest that REA combined with numerical analysis provides additional criteria and characteristic banding patterns for the study of the cross-contamination and characterization of C. chauvoei. The effects of temperature, oxygen tension, and enzymes on C. chauvoei hemolysin activity were also discussed. These microorganisms may be a potential contaminant of carcasses and widespread in soil of abattoir environments. The information of area-specific distribution of C. chauvoei strains and its toxin characteristics may give an efficient program in protecting cattle and other ruminants.

The karyotype of the long-finned pilot whale, Globicephala melaena.

The chromosomes obtained by culturing lymphocytes from blood samples of the long-finned pilot whale Globicephala melaena, were studied by Q- and C-banding. The culture method was very difficult to handle. The karyotype of the long-finned pilot whale has a typical cetacean 2n = 44 chromosome number. The O-band revealed a characteristic banding pattern, which showed a heteromorphism between individuals but not within individuals. These observations were supported by the C-banding, which disclosed clear size variation of heterochromatin in some chromosome pairs. A species specific C-band pattern was observed in comparison between Globicephala melaena and Globicephala macrorhynchus, the short-finned pilot whale. O- and C-band markers can be used in family- and population studies if a good technical quality can be achieved. The finding of an inactivation of one of the X-chromosomes in females opens the possibility of using the method of staining the sex chromatin to determine the sex of small foetuses (<12cm).

Genetic analysis of the Paramecium aurelia species complex (Protozoa: Ciliophora) by classical and molecular methods

The Paramecium aurelia complex includes 15 species (sibling species) and is characterised by inbreeding (to varying degrees in different species), causing an increase in intra‐specific differentiation. Investigations into inter‐ and intraspecific differentiation of strains originating from remote habitats within species of the complex were carried out by classical inter‐strain crosses and molecular analyses (RAPD‐PCR fingerprints, ARDRA riboprints, RFLP‐PCR analysis). RAPD analysis showed that all species in the complex possessed characteristic band patterns and the majority were also polymorphic intra‐specifically. A correlation exists between the degree of inbreeding characteristic for a species with differentiation of DNAgenotypes revealed by RAPD analysis within species, where inbreeders showed substantial variability of band patterns and moderate inbreeders were highly similar. RFLP analysis (a 480bp fragment of the gene coding the Hsp 70 protein) with the application of restriction enzyme TruII distinguished among species, while digestion with restriction enzyme AluI distinguished groups of species (clusters) and both enzymes revealed intra‐species polymorphism within P. dodecaurelia. ARDRA riboprinting (using a fragment of SSU‐LSU rDNA, about 2400 bp) with restriction enzymes HhaI, AluI, HinfI, TaqI distinguished groups of species with different band patterns. The majority of enzymes also demonstrated intra‐specific differentiation within P. dodecaurelia. TaqI also revealed intraspecific differences in P. biaurelia and P. tetraurelia. All species in the P. aurelia complex showed a high percentage of surviving hybrid clones in F 1 obtained by conjugation and F2 obtained by autogamy in inter‐strain crosses. A low percentage was observed only in F2 inter‐strain hybrids of P. tredecaurelia, however no cytological changes in the nuclear apparatuses were detected and similar band patterns existed in the studied strains. Future studies, including sequencing of rDNA fragments, may disclose deeper relationships of the species.

Atomic Force Microscopy Investigation of Vocal Fold Collagen

Collagen is an important constituent of the vocal fold extracellular matrix and is necessary for providing tensile strength and maintaining tissue geometry. Traditional investigations of vocal fold collagen using light and electron microscopy do not provide information on the organization and mechanical properties of collagen in native topographic state. The primary objective of this study was to use Atomic Force Microscopy (AFM) to examine the surface characteristics and organization of collagen in the deep layer of the lamina propria at nanoscale resolution. Experimental in vitro design. Freshly dissected porcine vocal folds were mounted on AFM discs and imaged under contact and tapping mode to obtain information on topographic distribution of collagen. AFM imaging of the deep layer of the lamina propria revealed dense, abundant collagen fibers with a characteristic banding pattern. The distribution of collagen was heterogeneous, with both hydrophilic and hydrophobic regions within a sample. AFM offers a useful tool to obtain topographic information about biologic samples at nanoscale resolution with minimal sample preparation. Mapping the topography and mechanical properties of vocal fold collagen is necessary for designing rheologically compatible bioimplants for the treatment of dysphonia resulting from vocal fold scarring and bowing.

Parental chromosome differentiation in citrangequat [ Fortunella sp. × ( Citrus sinensis × Poncirus trifoliata)] using CMA banding patterns

The efficiency of chromomycin A 3 (CMA) staining was examined for parental chromosome differentiation in citrange [ Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] and citrangequat ( Fortunella sp. × citrange). All of the accessions analyzed had the same chromosome number of 2n = 18. CMA staining revealed six characteristic banding patterns on the basis of the number and position of CMA positive bands (CMA +) as follows; A: two terminal and one proximal band, B: one terminal and one proximal band, C: two terminal bands, D: one terminal band, E: no band, and F: one proximal band. Chromosome CMA banding patterns of the accessions were 1A + 1B + 2C + 13D + 1F in Fortunella margarita, 2B + 2C + 7D + 7E in ‘Fukuhara’ orange, 2B + 10D + 6E in Poncirus trifoliata, 1B + 1C + 10D + 6E in citrange and 1A + 1C + 11D + 4E + 1F in citrangequat. The results of this study confirmed the intergeneric and tri-generic hybridity of citrange and citrangequat, respectively.

Specific cross-reactivity in sera from cystic echinococcosis patients in an enzyme-linked immunoelectrotransfer blot for cysticercosis diagnostics

A commercially available enzyme-linked immunoelectrotransfer blot originally intended for diagnosis of cysticercosis was evaluated for echinococcosis diagnosis, because a characteristic band pattern--different from the specific cysticercosis pattern--was observed in sera from patients with echinococcosis. This band pattern was observed in 29 (78%) of 37 parasitologically proven cystic echinococcosis patients. Specificity of these bands was 100% for echinococcosis, when tested with 75 control sera.

The IS6110 Repetitive DNA Element of Mycobacterium tuberculosis Is Not Detected in Exhaled Breath Condensate of Patients with Active Pulmonary Tuberculosis

Background: A large tertiary referral hospital in inner-city Chicago. Objectives: To determine whether the IS6110 repetitive DNA element of Mycobacterium tuberculosis is detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis. Methods: Ten hospitalized patients with positive Ziehl-Neelson-stained sputum smears were studied. Concurrent sputum cultures for mycobacteria were performed as well. Exhaled breath condensate was collected from each patient within 6 days of initiating antituberculosis chemotherapy (median 1.5 days). These samples were analyzed by polymerase chain reaction (PCR) using primers designed to amplify the IS6110 DNA fragment of M. tuberculosis. Exogenous M. tuberculosis DNA was added to exhaled breath condensate samples to detect PCR inhibitors. Concurrent cultures of exhaled breath condensate for mycobacteria were performed. Results:M. tuberculosis was identified in 9 of 10 sputum cultures. One isolate was identified as Mycobacterium kansasii. The IS6110 repetitive DNA element of M. tuberculosis was not detected in any of the 10 exhaled breath condensate samples. Exogenous M. tuberculosis DNA added to these samples elicited the characteristic band pattern of M. tuberculosis on agarose gel electrophoresis. No PCR inhibitors were detected. Cultures of exhaled breath condensate showed no growth of mycobacteria. Conclusions: The IS6110 repetitive DNA element of M. tuberculosis is not detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis.

A Molecular Fingerprint Technique to Detect Pollution‐Related Changes in River Cyanobacterial Diversity

Humans now have a strong influence on almost every major aquatic ecosystem, and our activities have dramatically altered the quality of receiving waters worldwide. Thus, there is a continuous need to develop and apply novel and effective technologies to detect, manage, and correct human-induced degradation of aquatic systems. In the present work, we evaluated the molecular approach using polymerase chain reaction (PCR)-temperature gradient gel electrophoresis (TGGE) to measure changes in cyanobacterial diversity along a pollution gradient in a river and compared it with that of using microscopic observations of field-fixed and cultured samples. The different 16S rDNA genes present in the cyanobacterial community of each sampling point of the river were separated by TGGE, giving a characteristic pattern of bands for each site. This pattern represents a "fingerprint" of the community, allowing direct comparisons of the different samples. The TGGE results revealed that the structure of the cyanobacterial community differed along the pollution gradient of the river. Microscopic and molecular approaches showed that cyanobacterial diversity decreased in a downstream direction. Similar results were obtained by the two methods, as indicated by the high correlation between them. We suggest PCR-TGGE could be a useful and rapidly applied technique for the routine analysis of changes in cyanobacterial diversity in response to pollution, which would allow us to monitor rivers in surveillance networks of watercourse quality.

Molecular mechanisms of paclitaxel and NM-3 on human gastric cancer in a severe combined immune deficiency mice orthotopic implantation model

To explore the molecular mechanisms of action of paclitaxel and NM-3 on human gastric cancer in severe combined immune deficiency (SCID) mice. Human gastric cancer cells SGC-7901 were implanted into SCID mice and mice were treated with paclitaxel and NM-3. The effects of paclitaxel and NM-3 on apoptosis of human gastric cancer cells were analyzed using flow cytometry, TUNEL assays, and DNA fragment analyses. Apoptosis of SGC-7901 cells was successfully induced by paclitaxel, NM-3, and the combination of paclitaxel and NM-3 24 h after injection as shown by the presence of apoptotic hypodiploid peaks on the flow cytometer before G1-S and a characteristic apoptotic band pattern in the DNA electrophoresis. The apoptotic rate detected by TUNEL assay was found to be significantly higher in the paclitaxel/NM-3 compared to the control group (38.5% +/- 5.14% vs 13.2% +/- 1.75%, P < 0.01). Paclitaxel in combination with NM-3 is able to induce apoptosis of the human gastric cancer cells in SCID mice effectively and synergistically.

Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity

To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. 8 naïve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.

Carbon nanotubes in an ancient Damascus sabre

The steel of Damascus blades, which were first encountered by the Crusaders when fighting against Muslims, had features not found in European steels--a characteristic wavy banding pattern known as damask, extraordinary mechanical properties, and an exceptionally sharp cutting edge. Here we use high-resolution transmission electron microscopy to examine a sample of Damascus sabre steel from the seventeenth century and find that it contains carbon nanotubes as well as cementite nanowires. This microstructure may offer insight into the beautiful banding pattern of the ultrahigh-carbon steel created from an ancient recipe that was lost long ago.

273 EXPRESSION PATTERN OF CONNEXIN 43 (Cx43) AND POLY-(A) POLYMERASE (PAP) GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED IN VITRO

Polyadenylation of pre-mRNA is carried out by poly-(A) polymerase (PAP), and study of the transcription pattern of this gene is said to indicate the developmental competence of the embryos. Connexin 43 (Cx43) is one of the important gap junction proteins that controls growth, cellular differentiation, and embryonic development. The objective of the present investigation was to study the expression pattern of PAP and Cx43 genes in buffalo (Bubalus bubalis) embryos produced in vitro. Embryos were produced from the slaughterhouse ovaries using standard IVMFC protocol (Rajhans et al. 2006 J. Reprod. Fertil. Dev. 18, 253–254). Briefly, oocytes were aspirated from follicles (2–8 mm in diameter) and matured in vitro in TCM-199 supplemented with 10% FCS and epidermal growth factor (20 ng mL-1) for 24 h. Presumptive zygotes after 18 h of fertilization were cultured in mSOF containing insulin-like growth factor-1 (100 ng mL-1) and β-mercaptoethanol (100 �M) for 9 days or until blastocyst formation, whichever was earlier. Pools of immature (n = 200), in vitro-matured (n = 200), oocytes and embryos (2–4 cell, n = 83; 8–16 cell, n = 80; morula, n = 77), and blastocysts (n = 40) were collected for mRNA isolation. Immature and in vitro-matured oocytes were treated with 1X trypsin-EDTA solution to remove the attached cumulus and then washed with TCM-199 before mRNA isolation to avoid any contamination of these cells during RNA isolation. mRNA from each pool was isolated using a commercially available direct mRNA isolation kit (Oligotex Direct mRNA kit; Qiagen, Valencia, CA, USA). cDNA was prepared using specific reverse primer and M-MLV RT in 20 �L reaction volume following manufacturer's instructions. Polymerase chain reaction was done for 35 cycles with annealing temperatures of 60�C and 58�C for 252 bp of PAP and 425 bp of Cx43, respectively. Amplicons were subjected to restriction endonuclease digestion for further confirmation of expressed genes. RT-PCR amplicon of PAP was digested with HaeIII to obtain characteristic band patterns at 119 bp and 133 bp, and Cx43 RT-PCR amplicon was digested with EcoR1 to obtain characteristic band patterns at 137 bp and 288 bp. While PAP expression could be detected in all stages of developing embryos starting from immature oocytes to blastocyst stage, Cx43 mRNA was detected in immature oocytes to morula stage but not in blastocyst-stage embryos. It could be concluded that the expression patterns of PAP and Cx43 genes in buffalo embryos produced in vitro are similar to those of cattle embryos.

Y-type gene specific markers for enhanced discrimination of high-molecular weight glutenin alleles at the Glu-B1 locus in hexaploid wheat

A number of primers were designed which target DNA sequence variation of the coding and /or promoter regions of wheat HMW glutenin y-type genes located at the Glu-B1 locus. This allowed the development of a set of PCR-based markers for specific HMW glutenin genes encoding By-subunits for which no markers were previously available. Markers were validated using test cultivars containing specific Glu-B1 alleles confirmed by SDS-PAGE and RP-HPLC analysis. Among the specific markers developed, primer pair ZSBy8F5/R5 was specific for the By8 gene, which exists in Glu-B1b (Bx7+By8) and Glu-B1u (Bx7*+By8) alleles. This marker allows discrimination of alleles containing By8 and By8* that are usually difficult to distinguish using SDS-PAGE. Since the over-expressed Glu-B1 allele ( Glu-Bl al.) contains the By8* subunit, it is possible to use this marker in breeding programs for selecting for the over-expression of subunit Bx7 in crosses that segregate between normal Bx7 and over-expressed Bx7 subunits. This marker also represents an alternative for distinguishing two common Glu-B1 alleles: Glu-B1i (Bx17+By18) and Glu-B1b (Bx7+By8). Two primer pairs ZSBy9aF1/R3 and ZSBy9F7/R6 both gave characteristic banding patterns for Glu-B1c (Bx7+By9) and can therefore be used to discriminate By9 - containing alleles from non - By9 alleles. Primer pair ZSBy9F2/R2 produced amplicons with a diagnostic banding pattern for allele Glu-B1f (Bx13+By16) and also permitted the discrimination of Glu-B1h (Bx14+By15) and Glu-B1e (Bx20) that have opposing genetic effects on wheat quality and are difficult to discriminate by SDS-PAGE.

Molecular Analysis (RAPD-PCR) of Inter-strain Hybrids of the Paramecium aurelia Species Complex (Ciliophora, Protozoa)

RAPD-PCR analysis showed that species of the Paramecium aurelia complex possessed characteristic band patterns and that the majority were also polymorphic intra-specifically. A comparison of band patterns was performed for some inter-strain hybrids within P. primaurelia, P. tetraurelia, P. pentaurelia, P. septaurelia, P. octaurelia, P. decaurelia, P. dodecaurelia, P. tredecaurelia, and P. quadecaurelia to band patterns characteristic for the parental strains. The investigations, however, did not reveal a close correlation between the degree of inbreeding characteristic for the species and similarity of genotypes. A low similarity of hybrid and parental band patterns was observed in P. octaurelia, P. dodecaurelia, P. quadecaurelia and also P. primaurelia. A high similarity of band patterns of hybrid and parental strains was found in P. tetraurelia, P. septaurelia, P. decaurelia, and P. tredecaurelia.

Karyotype and Banding Patterns of Chicken Breeds

3 Abstract: Traditional karyotyping is invented in animal research for several decades depend on the analysis of characteristic banding patterns along the length of chromosome. In the present study chicken metaphase chromosomes were obtained by peripheral blood lymphocyte culture techniques, G-band patterns were obtained with trypsin and Giemsa, C-band patterns were treated with barium and the nuclear organizer regions (NORs) were identified by silver staining. All species studied presented a diploid number of 7 8 chromosomes, with 10 pairs of macro chromosomes including the sex chromosome and 29 pairs of micro chromosomes. G-band patterns were found quite different between breeds. The dark stained of C-band was observed on micro chromosome and W chromosome. Karyotype resemblance near coefficient was possible for breeds clustering. The position of centromers, relative length, arm ratio and the evolutionary distance of chicken breeds was estimated. The application of chromosome karyotype and banding techniques was used to study the origin, evolution and relationship of species, also used for gene location and sex determination. While, in the Medical field was used to identify genetic disease. The techniques was consider as a base for further molecular research, for example FISH

Development of Hsp25 expression compartments is not constrained by Purkinje cell defects in the Lurcher mouse mutant

Four transverse zones can be distinguished in the adult mouse cerebellar cortex based on differential expression of cell-specific antigens, termination patterns of mossy fiber afferents, and phenotypes of mouse mutants with cerebellar defects: the anterior zone (AZ), central zone (CZ), posterior zone (PZ), and nodular zone (NZ). In the heterozygous Lurcher (Lc/+) mouse a zonally restricted abnormality in Purkinje cell development is seen. The Purkinje cell-specific antigen zebrin II is normally differentially expressed in all four zones of the adult cerebellum, but in the Lc/+ mutant is confined to the PZ and NZ, caudal to a transverse boundary in the dorsal aspect of lobule VIII. In this study we wanted to understand why zebrin II expression is arrested at this boundary and whether the Lc mutation affects the differentiation of additional Purkinje cell antigens in a similar manner. To determine this, we took advantage of the dynamic developmental timetable of another Purkinje cell antigen, the small heat shock protein Hsp25. Using immunohistochemistry we demonstrate that cerebellar maturation anterior to the CZ/PZ transverse boundary appears to be unaffected by the Lc allele, in that initial progression of Hsp25 expression in the Lc/+ cerebellum was similar to controls. Double-labeling experiments with anti-Hsp25 and anti-calbindin suggest that characteristic banding patterns of Hsp25 in Lc/+ cerebellum develop and are preserved despite cell loss. Thus, since simple temporal or spatial models cannot account for the zonal restriction seen during Lc/+ cerebellar development, the abnormality may be zebrin II-specific.