Inverse genetics tracing the differentiation pathway of human chondrocytes.

OBJECTIVE: Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process. DESIGN: The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway. RESULTS: We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPSCs. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9. CONCLUSIONS: This inverse genetic strategy may be useful for seeking candidates for the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported.

The absence of brain-specific link protein Bral2 in perineuronal nets hampers auditory temporal resolution and neural adaptation in mice.

Brain-specific link protein Bral2 represents a substantial component of perineuronal nets (PNNs) enwrapping neurons in the central nervous system. To elucidate the role of Bral2 in auditory signal processing, the hearing function in knockout Bral2(-/-) (KO) mice was investigated using behavioral and electrophysiological methods and compared with wild type Bral2(+/+) (WT) mice. The amplitudes of the acoustic startle reflex (ASR) and the efficiency of the prepulse inhibition of ASR (PPI of ASR), produced by prepulse noise stimulus or gap in continuous noise, was similar in 2-week-old WT and KO mice. Over the 2-month postnatal period the increase of ASR amplitudes was significantly more evident in WT mice than in KO mice. The efficiency of the PPI of ASR significantly increased in the 2-month postnatal period in WT mice, whereas in KO mice the PPI efficiency did not change. Hearing thresholds in 2-month-old WT mice, based on the auditory brainstem response (ABR) recordings, were significantly lower at high frequencies than in KO mice. However, amplitudes and peak latencies of individual waves of click-evoked ABR did not differ significantly between WT and KO mice. Temporal resolution and neural adaptation were significantly better in 2-month-old WT mice than in age-matched KO mice. These results support a hypothesis that the absence of perineuronal net formation at the end of the developmental period in the KO mice results in higher hearing threshold at high frequencies and weaker temporal resolution ability in adult KO animals compared to WT mice.

Optical imaging of mouse articular cartilage using the glycosaminoglycans binding property of fluorescent-labeled octaarginine.

OBJECTIVE: The aim of the current study was to examine the cartilage-specific binding property of polyarginine peptides (R4, 8, 12, and 16) and specifically to test octaarginine peptides for the optical imaging of articular cartilage in experimentally induced arthritis in mice. METHODS: Four rhodamine-labeled polyarginine peptides each with a different-length arginine chain (R4, 8, 12, or 16) were injected into the knee joints of C57BL/6J mice (n=20). The joints were excised 1h later and the fluorescent signal intensity in cartilage cryosections was compared for the four peptides. To examine the substrate of R8 in cartilage, femoral condyles obtained from another set of mice were treated with chondroitinase ABC (Ch'ase ABC), keratanase or heparitinase then immersed in R8-rhodamine. Fluorescent signals were examined by fluorescent microscopy. Next, R8-rhodamine was injected into the right knee joints of three control and three collagen antibody-induced arthritis (CAIA) mice, and fluorescent intensity in normal and degenerative cartilage was semi-quantitatively analysed on the histological sections using image software. Finally, femoral condyles from normal mice (n=2) and CAIA mice (n=2) were immersed in R8-rhodamine and calcein, then imaged using optical projection tomography (OPT). RESULTS: Fluorescent signals were specifically detected in the cartilage pericellular matrix from the surface to the tide mark but were completely absent in the calcified layer or bone marrow. The number of arginine residues significantly influenced peptide accumulation in articular cartilage, with R8 accumulating the most. The fluorescent signal in the femoral condylar cartilage diminished when it was treated with Ch'ase ABC. R8 accumulation was significantly decreased in the degenerative cartilage of CAIA mice, and this was demonstrated both histologically and in three-dimensional (3D)-reconstruction image by OPT. CONCLUSION: R8 may be a useful new experimental probe for optical imaging of normal and arthritic articular cartilage.

Determination of bovine serum low-density lipoprotein cholesterol using the N-geneous method.

The N-geneous method is a recently developed method for determination of low-density lipoprotein cholesterol (LDL-C) in human serum. In the present study, we attempted to adapt this method to bovine serum. The values of LDL-C obtained using the N-geneous method were highly correlated with those from the method using ultracentrifugation and heparin sepharose affinity chromatography (r = 0.934, p < 0.001). The reproducibility of this method was acceptable (intra-assay CV 4.2%, inter-assay CV 7.6%) for clinical use. Using the N-geneous method, serum LDL-C was evaluated in cows around parturition, and in cows with fatty liver induced by fasting. The concentration of LDL-C decreased significantly in cows close to parturition. A reduced concentration of LDL-C was also observed in cows with fatty liver. In both cases, the changes of LDL-C were similar to those of apolipoprotein B (apoB)-100, and the values of LDL-C were highly correlated (r = 0.876, p < 0.001) with those of apoB-100. These results suggest that the concentration of LDL-C reflects the level of apoB-100. The N-geneous method is simple and rapid, and might to be a useful tool to elucidate the clinical significance of LDL-C in bovine serum.

Distribution of apolipoprotein E among lipoprotein fractions in the lactating cow.

Apolipoprotein (apo) E plays a key role in regulating plasma levels of lipoproteins. We investigated the serum apoE concentrations in cows during different lactating stages by ELISA. To confirm the distribution of apoE in lipoprotein fractions, cow plasma was separated by gel filtration, ultracentrifugation and agarose gel electrophoresis. The apoE concentrations during early, mid- and late lactating stages in cows were significantly higher than that during the non-lactating stage. In lactating plasma, apoE eluted in high-density lipoprotein (HDL) fractions separated by gel filtration increased. The portion of this apoE in plasma was 49%. However, when lactating plasma was separated by ultracentrifugation, less then 5% apoE was recovered in the HDL fraction, and more apoE was recovered in the non-lipoprotein fraction (d>1.21 g/ml, 46%). In agarose gel electrophoresis, plasma apoE was found in beta-migrating lipoprotein, but it was not present in alpha-migrating lipoprotein. To purify apoE-containing particles, the HDL fraction separated by gel filtration was pooled and the fraction retained on Heparin-Sepharose chromatography collected. Cholesterol was absent from this fraction. These results suggest that apoE-containing particles, which increased during the lactating stage, were not associated with HDL particles, and that lipid-free forms were included in cow plasma.

Catecholamines and opioid peptides increase in plasma in humans during possession trances.

Naturally induced possession trances have been observed in healthy people of many societies. The neurophysiological basis of this phenomenon remains unknown, however, because of the difficulty in accessing subjects in trances due to their sacred context. In the present study, we measured the plasma levels of several neuroactive substances from subjects exhibiting or lacking possession trance characteristics during Balinese dedicatory dramas under natural conditions. The trance group exhibited significant increases in plasma concentrations of noradrenaline, dopamine and beta-endorphin, compared with controls who performed the same actions as the trance group. The present finding suggests that catecholamines and opioid peptides are involved in possession trances.

Neurocan is dispensable for brain development.

Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.

Increased serum concentration of apolipoprotein C-III and its greater distribution to chylomicrons than to the high-density lipoprotein fraction in a calf with hyperlipidemia.

A calf having extremely high concentrations of triglycerides, cholesterol and phospholipids, in particular in chylomicrons (CM) and very low-density lipoprotein (VLDL) fraction was found. The purpose of the present study was to determine serum concentration and distribution of apolipoprotein (apo) C-III, a low molecular mass protein mainly distributed in high-density lipoprotein (HDL) fraction in normolipidemic cattle, in the calf with hyperlipidemia. The serum apoC-III concentration in the calf increased to more than 10-fold that of normolipidemic control calves, and apoC-III was distributed more in the CM than in the HDL. The concentration of apoA-I (a predominant apoprotein in the HDL) was also increased to nearly 4-fold that of controls in the serum from the calf, and its major distribution site was the CM. Haptoglobin was detected in the serum from the hyperlipidemic calf, and was distributed in the CM as well as in the HDL. Serum amyloid A was also induced. In contrast to apoC-III, apoA-I and haptoglobin, the majority of apoSAA was found in the HDL fraction, as observed in normolipidemic calves. Increased concentrations in the CM of apoC-III and apoA-I suggest that the two apolipoproteins may be involved in the pathogenesis of calf hyperlipidemia. The presence of haptoglobin in the CM and HDL also implies the relevance of this acute-phase protein in the regulation of lipid metabolism.

The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain.

We report here molecular cloning and expression analysis of the gene for a novel human brain link protein-1 (BRAL1) which is predominantly expressed in brain. The predicted open reading frame of human brain link protein-1 encoded a polypeptide of 340 amino acids containing three protein modules, the immunoglobulin-like fold and proteoglycan tandem repeat 1 and 2 domains, with an estimated mass of 38 kDa. The brain link protein-1 mRNA was exclusively present in brain. When analyzed during mouse development, it was detected solely in the adult brain. Concomitant expression pattern of mRNAs for brain link protein-1 and various lectican proteoglycans in brain suggests a possibility that brain link protein-1 functions to stabilize the binding between hyaluronan and brevican. The human BRAL1 gene contained 7 exons and spanned approximately 6 kb. The entire immunoglobulin-like fold was encoded by a single exon and the proteoglycan tandem repeat 1 and 2 domains were encoded by a single and two exons, respectively. The deduced amino acid sequence of human brain link protein-1 exhibited 45% identity with human cartilage link protein-1 (CRTL1), previously reported as link protein to stabilize aggregates of aggrecan and hyaluronan in cartilage. These results suggest that brain link protein-1 may have distinct function from cartilage link protein-1 and play specific roles, especially in the adult brain.

Differential expression of mouse alpha5(IV) and alpha6(IV) collagen genes in epithelial basement membranes.

We first completed the primary structure of the mouse alpha5(IV) and alpha6(IV) chains, from which synthetic peptides were produced and a chain-specific monoclonal antibodies were raised. Expression of collagen IV genes in various basement membranes underlying specific organ epithelia was analyzed by immunohistochemical staining using these monoclonal antibodies and other antibodies from human and bovine sequences. It was possible to predict the presence of the three collagen IV molecules: [alpha1(IV)](2) alpha2(IV), alpha3(IV)alpha4(IV)alpha5(IV), and [alpha5(IV)](2)alpha6(IV). In skin basement membrane two of the three forms, [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV), were detected. The alpha3(IV)alpha4(IV)alpha5(IV) molecule was observed as the major form in glomerulus, alveolus, and choroid plexus, where basement membranes function as filtering units. The molecular form [alpha5(IV)](2)alpha6(IV) was present in basement membranes in tubular organs such as the epididymis, where the tubes need to expand in diameter. Thus, the distribution of the basement membranes with different molecular composition is consistent with tissue-specific function.

Inaudible high-frequency sounds affect brain activity: hypersonic effect.

Although it is generally accepted that humans cannot perceive sounds in the frequency range above 20 kHz, the question of whether the existence of such "inaudible" high-frequency components may affect the acoustic perception of audible sounds remains unanswered. In this study, we used noninvasive physiological measurements of brain responses to provide evidence that sounds containing high-frequency components (HFCs) above the audible range significantly affect the brain activity of listeners. We used the gamelan music of Bali, which is extremely rich in HFCs with a nonstationary structure, as a natural sound source, dividing it into two components: an audible low-frequency component (LFC) below 22 kHz and an HFC above 22 kHz. Brain electrical activity and regional cerebral blood flow (rCBF) were measured as markers of neuronal activity while subjects were exposed to sounds with various combinations of LFCs and HFCs. None of the subjects recognized the HFC as sound when it was presented alone. Nevertheless, the power spectra of the alpha frequency range of the spontaneous electroencephalogram (alpha-EEG) recorded from the occipital region increased with statistical significance when the subjects were exposed to sound containing both an HFC and an LFC, compared with an otherwise identical sound from which the HFC was removed (i.e., LFC alone). In contrast, compared with the baseline, no enhancement of alpha-EEG was evident when either an HFC or an LFC was presented separately. Positron emission tomography measurements revealed that, when an HFC and an LFC were presented together, the rCBF in the brain stem and the left thalamus increased significantly compared with a sound lacking the HFC above 22 kHz but that was otherwise identical. Simultaneous EEG measurements showed that the power of occipital alpha-EEGs correlated significantly with the rCBF in the left thalamus. Psychological evaluation indicated that the subjects felt the sound containing an HFC to be more pleasant than the same sound lacking an HFC. These results suggest the existence of a previously unrecognized response to complex sound containing particular types of high frequencies above the audible range. We term this phenomenon the "hypersonic effect."

Peri-implantation lethality in mice lacking the Sm motif-containing protein Lsm4.

Small nuclear ribonucleoproteins (snRNPs) are particles present only in eukaryotic cells. They are involved in a large variety of RNA maturation processes, most notably in pre-mRNA splicing. Several of the proteins typically found in snRNPs contain a sequence signature, the Sm domain, conserved from yeast to mammals. By using a promoter trap strategy to target actively transcribed loci in murine embryonic stem cells, a new murine gene encoding an Sm motif-containing protein was identified. Database searches revealed that it is the mouse orthologue of Lsm4p, a protein found in yeast and human cells and putatively associated with U6 snRNA. Introduction of the geo reporter gene cassette under the control of the murine Lsm4 (mLsm4) endogenous promoter showed that the gene was ubiquitously transcribed in embryonic and adult tissues. The insertion of the geo cassette disrupted the mLsm4 allele, and homozygosity for the mutation led to a recessive embryonic lethal phenotype. mLsm4-null zygotes survived to the blastocyst stages, implanted into the uterus, but died shortly thereafter. The early death of mLsm4p-null mice suggests that the role of mLsm4p in splicing is essential and cannot be compensated by other Lsm proteins.

Analysis of music-brain interaction with simultaneous measurement of regional cerebral blood flow and electroencephalogram beta rhythm in human subjects.

To elucidate the neural substrates of the receptive aspect of music, we measured regional cerebral blood flow (rCBF) with positron emission tomography (PET) and simultaneously recorded the electroencephalogram (EEG) in eight normal volunteers. Compared with the rest condition, listening to music caused a significant increase in EEG beta power spectrum (13-30 Hz) averaged over the posterior two third of the scalp. The averaged beta power spectrum was positively correlated with rCBF in the premotor cortex and adjacent prefrontal cortices bilaterally, the anterior portion of the precuneus and the anterior cingulate cortex in both the rest and the music conditions. Listening to music newly recruited the posterior portion of the precuneus bilaterally. This may reflect the interaction of the music with the cognitive processes, such as music-evoked memory recall or visual imagery.

The extracellular matrix in the mouse brain: its reactions to endo-alpha-N-acetylgalactosaminidase and certain other enzymes.

As our previous studies have indicated, the cingulate cortex of the adult mouse brain contains many neurons with rich cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The present study demonstrated that exclusive incubation with endo-alpha-N-acetylgalactosaminidase abolished the lectin Vicia villosa or Wisteria floribunda agglutinin (VVA or WFA) labeling of the nerve cell surface glycoproteins, while it neither interfered with the cationic iron colloid or aldehyde fuchsin stainings of the perineuronal proteoglycans nor abolished the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Double incubations with endo-alpha-N-acetylgalactosaminidase and collagenase did not eliminate the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, though they did eliminate the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans as well as the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Triple incubations with endo-alpha-N-acetylgalactosaminidase, collagenase, and endo-alpha-N-acetylgalactosaminidase abolished the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, and also eliminated the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans and the Gömöri's ammoniacal silver impregnation of the collagenous ligands. These findings indicate that: the nerve cell surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase; these galactosamines associated with the collagenous ligands or perineuronal proteoglycans are not digested by endo-alpha-N-acetylgalactosaminidase; and the terminal N-acetylgalactosamines newly exposed by collagenase incubation are digested by this galactosaminidase. It was further demonstrated that hyaluronidase incubation neither digests the collagenous ligands nor revives the lectin VVA or WFA labeling of the nerve cell surface proteoglycans.

Mouse ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues.

The Drosophila gene ten-m/odz is the only pair rule gene identified to date which is not a transcription factor. In an attempt to analyze the structure and the function of ten-m/odz in mouse, we isolated four murine ten-m cDNAs which code for proteins of 2,700-2, 800 amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain characteristic of transmembrane proteins, 300-400 amino acids after the NH2 terminus. About 200 amino acids COOH-terminal to this hydrophobic region are eight consecutive EGF-like domains. Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 showed that Ten-m1 is a disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5 which contain an odd number of cysteines. Northern blot and immunohistochemical analyses revealed widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

Differential tissular expression and localization of type IV collagen alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains and their mRNA in normal breast and in benign and malignant breast tumors.

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct alpha chains. We investigated the cellular regulation and origin of these alpha(IV) chains in normal and neoplastic breast tissues by immunohistochemistry by using alpha(IV) chain-specific antibodies and by in situ hybridization. In normal breast, alpha1(IV) and alpha2(IV) chains were stained in all BM, whereas alpha5(IV) and alpha6(IV) chains were restrictively localized in a linear pattern in the BM of the mammary gland. Similar immunostaining profiles were observed in benign breast tumors and in the intraductal components of invasive ductal carcinoma. However, in invasive ductal carcinoma, alpha1(IV) and alpha2(1V) chains were discontinuously or negatively stained in the cancer cell nests, and the assembly of alpha5(IV) and alpha6(IV) chains into the BM was completely inhibited. Coexpression of alpha5(IV) and alpha6(IV) chains was related to the localization of alpha-smooth muscle actin (alpha-SMA)-positive myoepithelial cells. By in situ hybridization, in fibroadenoma and invasive ductal carcinoma, the signals for alpha1(IV) and alpha2(IV) mRNA were abundant in stromal cells. However, the signals for alpha5(IV) and alpha6(IV) mRNA were not seen in any of these cells. In contrast, in intraductal papilloma, coexpression of alpha1 (IV)/alpha2(IV) mRNA and alpha5(IV)/alpha6(IV) mRNA was identified in epithelial cells. The results indicate that the mammary gland forms a second network of BM composed of alpha5(IV)/alpha6(IV) chains, in addition to the classic network of alpha1(IV)/alpha2(IV) chains. The expression of type IV collagen alpha chains seems to be differentially regulated by the epithelial-myoepithelial interaction and to be associated with the invasive potential of breast cancer.

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Differential expression of type IV collagen isoforms, alpha5(IV) and alpha6(IV) chains, in basement membranes surrounding smooth muscle cells.

Smooth muscle is composed of cigar-shaped, non-striated cells, each of which is encapsulated by a basement membrane and forms the contractile portion of tubular organs such as the gastrointestinal tract, pulmonary tract, genitourinary tract, and vasculature, in which slow and sustained contractions are needed. We examined basement membranes produced by smooth muscle cells and, using alpha(IV) chain-specific monoclonal antibodies, analyzed type IV collagens in these organs. Detailed distribution analysis of the alpha chains in normal and Alport cases by use of specific antibodies indicated that there are at least three molecular forms of type IV collagen, [alpha1(IV)]2alpha2(IV),alpha3(IV)alpha4(IV)alpha5+ ++(IV), and alpha5(IV)/alpha6(IV). Smooth muscle cells in the urinary bladder and uterus were enclosed by basement membranes composed of alpha1, alpha2, alpha5, and alpha6 chains. The same alpha chains were present around smooth muscle cells in the muscular layer of the fundus of the stomach, whereas those in the antrum and further distal side of the gastrointestinal tract expressed mostly alphal and alpha2 chains. In addition, immunostaining analysis of the vasculature also showed that most of the smooth muscle cells were positive for alpha1 and alpha2 chains; however, alpha5 and alpha6 chains were also expressed by smooth muscle cells in the aorta and some arteries where blood pressure changes significantly. These results suggest that the smooth muscle cells enclosed by alpha5/alpha6-containing basement membranes might have some particular function related to mechanical stress or tensile strength during the characteristic contractile activity of tubular organs.

Expression of type XVII collagen alpha 1 chain mRNA in the mouse heart.

The type XVII collagen alpha 1 chain has been identified as a component of the type I hemidesmosome, and is thus thought to play a role in extracellular matrix (ECM) maintenance and signal transduction between the cell and the ECM. We examined the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart by Northern blot analysis and determined the sequential changes of its expression in different developmental stages of the heart using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Northern blotting: Total RNA was extracted from 10 adult mouse hearts by the guanidine/cesium method. Hybridization was performed with mouse cDNA for alpha 1 (XVII) collagen. RT-PCR: Total RNA was extracted from 7 embryos, 4 neonates and 8 adult mice. Reverse transcription was performed using oligo-dT primer and MMLV. Amplification was carried out in alpha 1 (XVII) collagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control. Northern blotting revealed a 5.6 kb signal that was identical to that of the alpha 1 (XVII) of skin and transformed keratinocyte reported previously. The sequences of the PCR products were also identical to those reported. The normalized expression ratios of alpha 1 (XVII) were 0.91 +/- 0.20 in the embryonic heart, 0.36 +/- 0.20 in the neonatal heart and 0.96 +/- 0.21 in the adult heart. In conclusion, we identified the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart, suggesting that the type I hemidesmosome is located in the heart. The results of the RT-PCR at different developmental stages of the heart suggest that type XVII collagen contributes not only to cardiogenesis in the embryonic stage but also to maintenance of architecture and function in the adult heart.

Organization and expression of basement membrane collagen IV genes and their roles in human disorders.

Six distinct genes have been identified as belonging to the type IV collagen gene family. They can be organized into three sets, i.e., COL4A1/COL4A2, COL4A3/COL4A4, and COL4A5/COL4A6, which are localized on three different chromosomes in humans, 13, 2, and X, respectively. Within each set the genes are aligned head-to-head and their expression is regulated by bidirectional promoters between the genes. Transcriptional regulation of the COL4A1/COL4A2 set has been well characterized. The transcription of COL4A6 seems to be controlled by two alternative promoters. While collagen IV molecules composed of alpha1 and alpha2 chains are broadly distributed, molecules comprising combinations of the other four chains, alpha3-alpha6, are important components of specialized basement membranes. The precise chain composition of triple-helical molecules assembled from the alpha3-alpha6 chains is not entirely clear, but it is hypothesized that alpha3-alpha5 chains and alpha5 and alpha6 chains form heterotrimeric molecules. Several pieces of evidence indicate that alpha3/alpha4/alpha5 molecules and alpha5/alpha6 molecules are components of the basement membrane network. This helps explain the observation that the kidney and skin basement membranes from patients with Alport syndrome caused by mutations in the alpha5 coding gene, COL4A5, are defective in the alpha3, alpha4, and alpha6 chains together with the alpha5 chain. Large deletions involving the COL4A5 and COL4A6 genes have been found in rare cases of diffuse leiomyomatosis associated with Alport syndrome.