Expression of hsc 70, but not hsp 70, in human third molar dental pulp.

The constitutive (hsc 70) and inducible (hsp 70) isoforms of heat shock protein 70 are important members of the superfamily of stress related proteins that protect and promote the recovery of cells from physiological and pathologic stress. The goal of this study was to define the baseline expression of hsc 70 and hsp 70 in disease-free, minimally stressed human dental pulp of the adult 3rd molar. Immunolocalization demonstrated moderate to heavy staining intensity for hsc 70 in both the cytoplasm and nucleus of odontoblasts and fibroblasts comprising the human pulp. Endothelial and smooth muscle cells displayed weak to moderate immunoreactivity for hsc 70 in both the cytoplasm and nucleus. Schwann cells demonstrated only weak nuclear staining for hsc 70. No immunoreactivity for hsp 70 was observed in any cell type in human pulp. Western, northern, and RT-PCR analysis of pulp preparations confirmed the expression of hsc 70 mRNA and protein within components of the pulp. These results demonstrate that cells of the human pulp express, under conditions of minimal stimulation, a key component of the stress response protein superfamily. The expression of hsc 70 under conditions of minimal stress may provide pulp components an advantage in resisting cell injury when stress occurs.

Expression of heat shock protein 27 in adult human third molar dental pulp.

This study is the first to define the expression of hsp 27 in the pulp of the adult human third molar. Using a monoclonal antibody against human hsp 27, immunoreactivity was demonstrated in the odontoblasts, odontoblast processes, pulp fibroblasts, and smooth muscle and endothelial cells of vessel walls. Nerves were negative. Pulp fibroblasts were characterized by cytoplasmic staining and variable nuclear staining. Odontoblasts also displayed consistent cytoplasmic staining and variable nuclear staining. Western, Northern, and RT-PCR analysis confirmed the expression of hsp 27 mRNA and protein. Hsp 27 was also shown to be present in both the unphosphorylated and phosphorylated isoforms. In general, nuclear localization and phosphorylation of hsp 27 has been correlated with cells responding to stress or other stimuli. This study demonstrates that pulp from a single human third molar provides sufficient material to support a detailed molecular analysis of gene expression.

Isoform-specific expression of metallothionein mRNA in the developing and adult human kidney.

The organization of the metallothionein (MT) gene family has been demonstrated to be much more complex in humans than in the mouse, and possibly rodents in general. For humans, the MTs are encoded by a family of genes located at 16q13 representing 10 functional and 7 non-functional MT isoforms. In the present study, the 5' and 3' untranslated region sequences of the highly conserved, functional MT genes were utilized to generate primer pairs for the analysis of isoform-specific MT mRNA using reverse transcriptase-polymerase chain reaction (RT-PCR). Human kidneys from 13 weeks gestation through adulthood were examined for the expression of MT protein and mRNA. Immunohistochemical analysis demonstrated MT immunoreactivity to be confined exclusively to the proximal tubules of the adult and developing kidney. For all MT-positive cells, MT was localized in the cytoplasm and nuclear localization was variable. There was no correlation between nuclear staining and stage of development. Of the 10 MT genes examined (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-2A, MT-3, and MT-4), mRNAs representing the MT-1E, MT-1F, MT-1X, and MT-2A genes were consistently expressed in all samples regardless of gestational age. There was no indication of a 'fetal form' of MT analogous to that noted to occur in human liver. Messenger RNA for the MT-1A gene was detected in 2 of 6 renal samples without correlation to gestational age. In no instance was mRNA for the MT-1B, MT-1G, MT-1H, MT-3 or MT-4 genes detected. These studies detail the initial determination of MT gene expression in the human renal system and provide the PCR primers for testing and determination of MT gene expression in other organ systems.

Expression of heat shock protein 27 in developing and adult human kidney.

The expression of heat shock protein (hsp) 27 was examined in the developing and adult human kidney. Immunolocalization using a monoclonal antibody against human hsp 27 demonstrated immunoreactivity in both the developing and adult kidney. Low to moderate immunoreactivity for hsp 27 was observed in the fetal and adult proximal tubule, distal tubule, and mesangial cells of the glomeruli. Intense immunoreactivity for hsp 27 was localized to the cortical and medullary collecting ducts in both the adult and fetal kidney, with the most intense staining in the medullary regions. The loop of Henle demonstrated no immunoreactivity for hsp 27. The blastemal element of the developing kidney showed no hsp immunostaining and the ureteric bud demonstrated moderate staining. Western, northern, and reverse transcription-polymerase chain reaction (RT-PCR) analyses disclosed no significant differences in hsp 27 mRNA or protein level as a function of gestational age. An analysis of the phosphorylation state of hsp 27 showed the majority of hsp 27 to be present in the unphosphorylated isoform for both adult and fetal samples. These studies are the first to demonstrate the presence of hsp 27 in the human kidney. It is suggested that this pool of hsp 27 is constitutive as it appears in an inactivated state; localized to the cytoplasm and in an unphosphorylated state.

Development of cochlear potentials in the neonatal gerbil.

The onset and maturation of hearing was examined in separate groups of sibling and nonsibling neonatal Mongolian gerbils (Meriones unguiculatus). Auditory nerve compound action potentials (CAP) and cochlear microphonics (CM) were measured at the round window, and the endocochlear potential (EP) was recorded at three different locations in pups aged 13 to 30 days after birth (DAB) and in 90 day-old animals. Maturational trends for the three potentials were similar to those previously reported for gerbil neonates. However, CAP thresholds continued to decrease, and CM and CAP input/output functions and EP continued to increase beyond 30 days of age, a time at which many investigators have considered hearing in the gerbil to be mature. The EP developed simultaneously throughout the cochlea and approached 80 mV by 20 DAB. CAP thresholds showed a highly correlated log-linear relationship with EP in groups of nonlittermates and in siblings studied at different ages. In contrast, maximum CAP and CM amplitudes increased with increasing EP, but did not show significant growth until the EP exceeded 70 mV.

Distribution of immunoreactive alpha- and beta-subunit isoforms of Na,K-ATPase in the gerbil inner ear.

Biochemical and histochemical studies have demonstrated abundant Na,K-ATPase in the inner ear and provided new information concerning the ion transport capacities of specialized cell types. To extend these earlier observations, we immunostained inner ears from adult gerbils with antibodies specific for the three known alpha- and the two known beta-isoforms of Na,K-ATPase. Different inner ear cell types contained specific and distinct combinations of alpha- and beta-subunit isoforms. Strial marginal cells and vestibular dark cells expressed the alpha 1- and beta 2-isoforms, whereas other positive epithelial cells expressed alpha 1 in combination with beta 1. Ganglion neurons and their peripheral processes showed positive immunostaining for the alpha 3- and beta 1-subunit isoforms. Subpopulations of fibrocytes in the spiral prominence, suprastrial and supralimbal regions, and vestibular system expressed either the alpha 1- or alpha 2-isoform, or both. The differential expression of Na,K-ATPase subunit isoforms presumably reflects different K+ and Na+ transport capacities among inner ear cell types which, working in concert, serve to generate and maintain the unique ionic and electrical environment in the mammalian inner ear.