Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis.

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.

Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal epithelium of submandibular gland.

In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some "pioneer cells," rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.

Tumor progression in hepatocellular carcinoma may be mediated by p53 mutation.

Human hepatocellular carcinoma (HCC) often contains intratumoral subpopulations of heterogeneous cellular differentiations within each tumor. To analyze the genetic alterations of p53 in the heterogeneous subpopulations, we examined 68 intratumoral nodular lesions within 34 HCCs composed of two distinct subpopulations. The cellular differentiations were determined histologically by Edmondson's grading system. Nine (26.5%) of 34 HCCs examined were found to have genetic alterations in exons 5 to 8 of the p53 gene, resulting in amino acid substitutions. Three of these nine HCCs with p53 mutations showed genetic heterogeneity of the p53 gene within each tumor; one HCC had a single missense mutation at codon 210 (asparginine to 210-serine) in an intratumoral lesion of Edmondson Grade II and double missense mutations at codons 210 and 217 (asparginine to 210-serine and valine to 217-alanine) in another intratumoral lesion of Edmondson Grade III. The remaining two HCCs had p53 mutations only in lesions of a higher grade. In total, the p53 mutations were detected in none of eight Edmondson Grade I lesions, in five of 29 Grade III lesions (17.2%), in eight of 26 Grade III lesions (30.8%), and in three of five Grade IV lesions (60.0%). Thus, our data revealed that the p53 mutations were closely related to the progression of HCC and that, in certain cases, malignant cells which acquired the p53 mutations might develop into dedifferentiated subpopulations within individual HCC.

Basement-membrane stromal relationships: interactions between collagen fibrils and the lamina densa.

Collagens, the most abundant molecules in the extracellular space, predominantly form either fibrillar or sheet-like structures-the two major supramolecular conformations that maintain tissue integrity. In connective tissues, other than cartilage, collagen fibrils are mainly composed of collagens I, III, and V at different molecular ratios, exhibiting a D-periodic banding pattern, with diameters ranging from 30 to 150 nm, that can form a coarse network in the extracellular matrix in comparison with a fine meshwork of lamina densa. The lamina densa represents a stable sheet-like meshwork composed of collagen IV, laminin, nidogen, and perlecan compartmentalizing tissue from one another. We hypothesize that the interactions between collagen fibrils and the lamina densa are crucial for maintaining tissue-tissue interactions. A detailed analysis of these interactions forms the basis of this review article. Here, we demonstrate that there is a direct connection between collagen fibrils and the lamina densa and propose that collagen V may play a crucial role in this connection. Collagen V might also be involved in regulation of collagen fibril diameter and anchoring of epithelia to underlying connective tissues.

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model.

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 microg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 microm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 microg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along the dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.

Collagen II containing a Cys substitution for Arg-alpha1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils.

A recombinant human procollagen II was prepared that contained a substitution of Cys for Arg at alpha1-519 and that was found in five families with early onset generalized osteoarthritis with or without features of a mild chondrodysplasia. Previously, the presence of mutated monomers in mixtures with wildtype collagen II was shown to increase the lag period for fibril assembly. Also, the fibrils were more loosely packed and some thick fibrils lacked a D-periodic banding pattern. Here we re-examined the fibrils using a combination of transmission electron microscopy and atomic force microscopy. The presence of the mutated monomers increased the diameter of the thin filaments that were consistently formed in association with the thick fibrils of collagen II. In addition, the presence of the mutated monomers increased the depth of the gap regions in all fibrils with a distinct D-periodic banding pattern. The results, therefore, may indicate that the mutated monomers formed two or three additional outer layers of monomers in 0D-period staggers on the surface of the fibrils. Apparently, the mutated monomers were bound on the surface through intermolecular disulfide bonds.

Immunohistochemical distribution of calcium-activated neutral proteinases and endogenous CANP inhibitor in the rabbit hippocampus.

Intracellular accumulation of Ca2+ after brain ischemia is regarded as one of the principal causes of neuronal death, but details of the intracellular events occurring after Ca2+ accumulation have not yet been described. We propose that a calcium-activated neutral proteinase which can degrade neuronal cytoskeletal proteins might link Ca2+ accumulation and irreversible injury of the neuronal intracellular structure. First, therefore, we examined the distribution of calcium-activated neutral proteinase in normal brains. Immunohistochemical distribution of calcium-activated neutral proteinases (CANP) with high and low sensitivity to Ca2+ (muCANP and mCANP) and of endogenous CANP inhibitor was investigated in the dorsal hippocampus of the rabbit. muCANP-immunoreactivity was detected in almost all of the pyramidal cells and granule cells and in some other neurons. A full-length staining from perikarya to dendrites was shown in muCANP-positive neurons. mCANP-immunoreactivity was found mainly in four kinds of hippocampal interneurons: 1) basket cells in the stratum oriens of Ammon's horn, 2) pyramidal basket cells at the boundary of pyramidal cell layer and stratum oriens, 3) polymorphic cells in the hilar region of dentate gyrus, and 4) pyramidal or fusiform basket cells at the inner boundary of the granule cell layer and the hilar region. The distribution of these four kinds of neurons was similar to that of parvalbumin-containing GABAergic neurons. CANP inhibitor immunoreactivity was confined to pyramidal cells in the CA3-CA3c region and some hilar neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of collagen IV in acutely deteriorated renal allografts.

BACKGROUND: The changes in the basement membrane occurring in acutely deteriorated renal allografts (ADR) have not been extensively investigated. Our purpose is to elucidate the alteration of collagen IV, a main constituent of the basement membrane in ADR. METHODS: Fifty biopsy specimens of ADR and 10 of chronic transplant nephropathy (CTN) were examined with two monoclonal antibodies specific for collagen IV. JK199 and JK132 are monoclonal antibodies that recognize triple helical collagen IV containing the alpha1 chain. JK199 recognizes all the basement membrane containing [alpha1 (IV)]2alpha2(IV), although JK132 reacts only with a limited portion of it. In the normal kidney, JK199 reacts with the mesangial matrix, the basement membrane of Bowman's capsule (BBM), and the tubular basement membrane, as well as with the glomelular basement membrane (GBM). JK132 reacts with the mesangial matrix, BBM, and the tubular basement membrane. RESULTS: In ADR, increased intensity of JK199 was observed in GBM, the mesangial matrix, BBM, the tubular basement membrane, and the interstitium. Increased intensity of JK132 was observed in the mesangial matrix, BBM, and the tubular basement membrane, but was not remarkable in GBM or the interstitium. In contrast, biopsy specimens of CTN showed increased intensity of JK132 in GBM, the mesangial matrix, BBM, the tubular basement membrane and the interstitium. CONCLUSION: These results suggest that collagen IV is up-regulated in ADR. Differential staining of collagen IV with JK199 and JK132 in GBM and the interstitium may contribute to diagnose CTN.

Combined hepatocellular and cholangiocarcinoma: proposed criteria according to cytokeratin expression and analysis of clinicopathologic features.

We herein evaluated 36 cases of combined hepatocellular and cholangiocarcinoma (cHCC-CC) (including 29 surgically resected and seven autopsy cases) by the immunohistochemical methods of anticytokeratin antibodies 7 and 19, and then analyzed the clinicopathologic features by comparing cHCC-CC with ordinary hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The results indicated that even if mucin production could not be confirmed, nine cases with HCC areas that showed a histological resemblance to CC also showed immunohistological biliary differentiation. Therefore, we advocate that these HCC with biliary differentiation based on an immunohistochemical analysis should thus be included in the criteria of cHCC-CC in broad terms. Regardless of the extent of mucin production, the cHCC-CCs as indicated by an immunohistochemical analysis are considered to have a similar background to that of ordinary HCCs regarding such factors as the average age, male:female ratio, hepatitis B surface antigen (HBsAg) and hepatitis C virus antibody (HCVAb) positivity, alpha-fetoprotein level, and the presence of cirrhosis. However, cHCC-CCs tend to metastasize to many organs and the lymph nodes, and, as a result, have a poor prognosis.

The importance of laminin 5 in the dermal-epidermal basement membrane.

The skin consists of two main layers, epidermis and dermis, separated by the basement membrane. Epidermal-dermal communication through the basement membrane is important for skin homeostasis. The basement membrane contains specialized structures, called the anchoring complex, which ensure the stability of connection and communication between these two tissue compartments. The proteins within the anchoring complex provide links to both the intracellular cytoskeletal keratins in keratinocytes and connective tissue proteins of the dermis. One of the key components of the complex is laminin 5, which is essential to epidermal cell attachment. The biological function of laminin 5 has been investigated by using a skin equivalent model in vitro and during keratinocyte sheet grafting in vivo. As a major link between the epidermal basal cells and the papillary dermis, laminin 5 initiates hemidesmosome formation and provides stable attachment of the epidermis to the dermis. Laminin 5 also accelerates the assembly of basement membranes and may enhance the recovery of damaged skin. An intact basement membrane at the epidermal-dermal junction is essential to stability of the skin.

Gelation of the lens capsule type IV collagen solution at a neutral pH.

It is known that the type IV collagen extracted from EHS tumor assembles under a physiological condition, but not in a gel form. The EHS type IV collagen requires the other basement membrane components, laminin1, heparansulfate proteoglycan, and/or nidogen for gelation. On the other hand, Muraoka et al. reported that the bovine lens capsule type IV collagen alone gelated under a unique and unexpected condition of 2 M guanidine-HCl and 50 mM dithiothreitol, a condition which is thought to be dissociative for most biological macromolecules, including extracellular matrix [Muraoka, M. et al. (1996) J. Biochem. 119, 167-172]. The present report shows that the bovine lens capsule type IV collagen formed a gel under physiological conditions of pH and ionic environment, though the apparent rigidity of the gel was weaker than that of the gel formed in 2 M guanidine-HCl and dithiothreitol. The rigidity depended greatly on the incubation temperature and NaCl concentration of the type IV collagen solution, as observed in terms of the contractility of gel volume under centrifugal force. the gel formed in 150 mM NaCl and 20 mM phosphate, pH 7.3, at 28 degrees C contracted to 20% of the original volume on centrifugation of 1,800 x g for 10 min, while the gel formed at 4 degrees C, where type I collagen did not gelate at all, retained 90% of the original volume at the same centrifugal force. NaCl concentration was another important factor influencing the mechanical properties of type IV collagen gel. The gel formed at 150 mM showed maximal rigidity in the range of 0 to 300 mM in terms of the contractility on centrifugation. An image of a Pt/C replica of the gelated type IV collagen reconstituted at 4 or 28 degrees C in 20 mM phosphate, pH 7.3, containing 150 mM NaCl showed fine meshworks consisting of rather homogeneous pore sizes, resembling the skeletal structure of basal lamina. Since the condition where the type IV collagen alone formed gels was physiological in terms of ionic strength and pH, the aggregate structure and gel properties might reflect the in vivo type IV collagen supramolecular structure and the property.

Condensation of collagen fibrils to the direct vicinity of fibroblasts as a cause of gel contraction.

Fibroblasts were cultured within FITC-prelabeled type I collagen. Cells initially round in shape protruded processes, and began to collect the fibrils into their vicinity. Repeated protrusion and withdrawal of cell processes was observed. Consequently, condensed fluorescence was observed on the elongated bipolar cells stained with rhodamine-phalloidin. Concomitantly with these events, the gel began to contract in overall size, with an increase of fluorescence intensity. Scanning electron micrographs of the contracted gel showed a disproportional distribution of collagen fibrils: a highly condensed region surrounding cell bodies and a moderately condensed region. A major portion of condensed fibrils may have been derived from reconstituted collagen fibrils, since fibroblasts within collagen gel synthesized little collagen. When the gel adhered to glass tightly, so that overall contraction was prevented, the fluorescence in a range of scores of micrometers from the cells disappeared owing to depletion of fibrils by the cells. The combined spaces with null fluorescence in total under repressed contraction corresponded well to the reduction in volume due to gel contraction. It seems likely that the fibril condensation onto the cells causes the overall gel contraction.

Characterization of a monoclonal antibody against human placenta type IV collagen by immunoelectroblotting, antibody-coupled affinity chromatography, and immunohistochemical localization.

We have produced four monoclonal antibodies against type IV collagen obtained from human placenta. An antibody with a high titer by ELISA, named JK-199, reacted not only with type IV collagen in the triple-helical conformation but also with thermally denatured chains. After affinity chromatography on JK-199 antibody-coupled resin, the amino acid composition and CD spectrum of the affinity-purified peptides from the crude pepsin extract of human placenta were typical of those of human type IV collagen in the triple-helical conformation. On SDS-polyacrylamide gel electrophoresis, the purified protein showed only one broad band with a molecular weight of approximately 260,000 before reduction and six smaller peptide bands after reduction. On immunoelectroblotting, JK-199 reacted with all six peptide bands. Immunohistochemically, typical basement membranes were exclusively and strongly stained with JK-199 on frozen sections of PLP-fixed human placentas without any enzymatic pretreatment in the routine immunoperoxidase method. Judging from these findings, it is concluded that the epitopes of type IV collagen that reacted with JK-199 are exposed on the surface of basement membranes. This antibody should be useful for identification of type IV collagen in normal or pathological basement membranes or other structures.

Interaction of collagen molecules from the aspect of fibril formation: acid-soluble, alkali-treated, and MMP1-digested fragments of type I collagen.

Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1 (MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37 degrees C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30 degrees C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation.

Alkali-treated collagen retained the triple helical conformation and the ligand activity for the cell adhesion via alpha2beta1 integrin.

Alkaline treatment is a good method for extracting collagen with high recovery even from an aged animal specimen. However, the properties of collagen treated under alkaline conditions have not been well established yet. By the treatment with a solution of 3% sodium hydroxide and 1.9% monomethylamine, the isoelectric point of type I collagen was lowered from 9.3 to 4.8 because of the conversions of Asn and Gln to Asp and Glu. With the acidification of the pI, the denaturation temperature of the collagen was decreased from 42 to 35 degrees C after 20 d treatment, but the collagen-specific triple helical conformation was maintained. Human keratinocytes and fibroblasts adhered to the alkali-treated collagen via the collagen receptor integrin alpha2beta1. This indicates that the alkali-treated collagen maintained its property as a biological adherent molecule. Unlike acid-soluble collagen, alkali-treated collagen lost the ability to form fibrils at neutral pH under physiological conditions. This ability was lost even after 4 h of alkaline treatment, when the denaturation temperature of the collagen did not change. On the other hand, the alkali-treated collagen formed a fibrous precipitate with a uniform diameter of 50-70 nm under acidic conditions at 30 degrees C.

Localization of human placental glucose transporter 1 during pregnancy. An immunohistochemical study.

To elucidate the potential roles of glucose transporter 1 (GLUT1) in human placenta during pregnancy, we examined the localization of GLUT1 in human placenta at various stages by immunohistochemistry with an anti-GLUT1 antibody by use of both light and electron microscopy. Specific staining for GLUT1 was localized on the apical brush border and along the basal plasma membrane of the syncytiotrophoblasts. The staining at the apical side was more intense than that at the basal side during the early stages of gestation. In later gestational stages, however, the staining pattern at the apical side became blurred and the staining intensity at the basal side increased. The cytotrophoblasts, seen embedded in the basal part of the syncytiotrophoblasts, seemed to show immunoreactivity for GLUT1 along the plasma membranes at the light-microscopic level. However, immuno-electron microscopic analysis with either pre- or post-embedding methods revealed that specific staining for GLUT1 was hardly observed on the cytotrophoblasts, but the cytotrophoblasts were often surrounded by immunoreactive processes of syncytiotrophoblasts. The blood capillaries and erythrocytes in the stroma of placental villi were always immunoreactive for GLUT1 throughout pregnancy. These findings suggest that GLUT1 may play a vital role in human pregnancy.

Liver resection for hepatocellular carcinoma in the elderly.

OBJECTIVE: To estimate the effectiveness of hepatic resection on hepatocellular carcinoma (HCC) in the elderly. DESIGN: Comparison with younger patients. SETTING: A municipal hospital and a large university hospital in Japan. PATIENTS: The study included 39 patients (age > or = 70 years [the elderly group]) and 229 patients (age < 70 years [the younger group]) who underwent hepatic resection from April 1985 to March 1993. The preoperative clinical features (Child's classification, association of cirrhosis and liver functions) were comparable between two groups. The positive rate for hepatitis C virus antibody was higher in the elderly group (88% vs 59%; P = .016). MAIN OUTCOME MEASURES: Morbidity and survival following operation and the pathological features of HCC. RESULTS: The incidence of postoperative hepatic failure was higher in the elderly group (10% vs 2%; P = .018). However, the incidence of operative death in the elderly group (5% vs 1%) as well as the incidence of other postoperative complications and rates of long-term survival (75.9% vs 51.6% at 5 years) and disease-free survival (30.4% vs 31.0% at 5 years) were similar to those in the younger group. The pathological features of HCC were identical between the two groups. CONCLUSION: The outcome of surgical treatment of HCC in the elderly group was satisfactory when compared with that in the younger group.

The effects of vasodilating drugs on pH in tumors.

Hydralazine has been used widely to reduce tumor blood flow and thereby to induce hypoxia and to reduce extracellular pH (pHe) in tumors. Here we have investigated and compared the effects of the vasodilating drugs hydralazine, captopril, nifedipine, prazosin, sodium nitroprusside, and labetalol to reduce pHe in EMT-6 and KHT tumors of mice and to cause antitumor effects. After a single injection, captopril was most effective in reducing pHe in EMT-6 tumors with a decrease in mean pHe from 6.93 to 6.67 at 2 h after injection, while nifedipine was most effective for KHT tumors with a decrease in mean pHe from 6.96 to 6.75 at 1 h after injection. During 72 h of chronic administration into mice bearing tumors, nifedipine was ineffective in reducing pHe, but both captopril and hydralazine caused a small but significant reduction of pHe. Captopril caused significant delay in growth of the tumors, but had only a small effect on clonogenic cell survival. Captopril appears to be the most effective vasodilating drug to enhance tumor acidity.